Device for particle manipulation

ABSTRACT

An operation pipe and a device equipped with the operation pipe, which use a gel to perform operations such as separation, extraction, purification, elution, recovery, analysis and the like of target components that are biological components such as nucleic acids. More specifically, an operation pipe and a device, with which it is possible to perform operations such as separation, extraction, purification, elution, recovery, analysis and the like of target components in a sealable pipe by operating magnetic particles in the pipe under a magnetic field from outside of the pipe.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority benefit of Japan application serialno. 2019-043228, filed on Mar. 9, 2019. The entirety of theabove-mentioned patent application is hereby incorporated by referenceherein and made a part of this specification.

BACKGROUND OF THE DISCLOSURE Technical Field

The disclosure relates to an operation pipe and a device equipped withthe operation pipe, which use a gel to perform operations such asseparation, extraction, purification, elution, recovery, analysis andthe like of target components that are biological components such asnucleic acids. More specifically, the disclosure relates to an operationpipe and a device, with which it is possible to perform operations suchas separation, extraction, purification, elution, recovery, analysis andthe like of target components in a sealable pipe by operating magneticparticles in the pipe under a magnetic field from outside of the pipe.

Related Art

By giving various chemical affinity to surfaces of water-insoluble fineparticles having a diameter of 0.5 am to ten-odd micrometres, the targetcomponents can be separated, extracted, purified, eluted, recovered andthe like. In addition, target cells can also be recovered by recognizingspecific cell surface molecules. Based on these findings, fine particlesin which functional molecules having affinity for the target componentsare introduced onto the particle surface are commercially available.Among these fine particles, ferromagnets of which the particle materialis iron oxide or the like can recover the target components by a magnet,and have a feature advantageous for automation of the extraction andpurification of the target components because centrifugation is notrequired.

For example, a system for continuously performing nucleic acidextraction from cells to analysis performed by gene amplificationreaction in one device is commercially available. For example, inGeneXpert System (non-patent literature 1) of Cepheid, USA, the nucleicacid extraction to the analysis performed by gene amplification reactionare performed in one cartridge-type device, and the maximum number ofspecimens simultaneously processed is 16. In addition, for example, inSimplexa (non-patent literature 2) of 3M Corporation, the nucleic acidextraction to PCR can be performed in one disk-shaped device, and 12specimens can be fixed on one disk. However, these devices have a smallnumber of specimens simultaneously processed, and the devices themselvesare complicated and expensive to manufacture, making the devicesimpractical. In addition, the device size is large and thus the entiresystem is also large, making the devices impractical in terms ofmobility from an installation location.

Magnetic particles are commercially available as a part of reagents thatare extraction and purification kits. The kit is configured by aplurality of reagents contained in separate containers, and the usercollects and dispenses the reagents by a pipette or the like during use.Even in a case of an automated device, in a currently marketed device,liquid collection is mechanically performed by pipetting operations. Forexample, a system (non-patent literature 3) for performing nucleic acidextraction using magnetic particles is commercially available fromPrecision System Science Co., Ltd. These pipetting operations areaccompanied by generation of aerosols. The generation of aerosolincreases the risk of contamination that hinders analysis. The same alsoapplies to a case in which the liquid collection is mechanicallyperformed by pipetting operations in an automated device. In this case,since pollution source is accumulated in the device due to thegeneration of aerosols, it is necessary to periodically clean thedevice. However, in the device automated by a pipette-type dispensingmechanism, the structure is complicated, and it is difficult tocompletely remove the pollution source.

In general, the commercially available magnetic particles enableseparation and recovery of target components or specific cells from asample, but it is necessary to perform analysis of the recoveredmaterial in another system such as a real-time PCR device, a massspectrometer, a flow cytometry or the like. In the system which iscommercially available from Precision System Science Co., Ltd for usingmagnetic particles to perform nucleic acid extraction, even the recoveryof purified nucleic acids can be performed, but it is necessary toperform the analysis performed by gene amplification reaction and thelike in another system such as a real-time PCR device or the like.Furthermore, in this system, the dispensing using a pipette-typedispenser is performed in an open system, and thus is always accompaniedby a risk of contamination.

In order to solve the above problems, an operation pipe that is smalland has low running cost and a device equipped with the operation pipeare reported, with which it is possible to perform extraction andpurification of target components in a completely sealed container whileavoiding contamination or possible to analyze the target components inthe same container while keeping the sealed state following theextraction and purification (patent literature 1). A sealable narrowpipe constituting the operation pipe is filled with one or more liquidreagents partitioned by a water-insoluble gel substance without using adispenser accompanied by the generation of aerosols, and the magneticparticles present in the liquid reagents filled in the narrow pipe aremoved by a magnetic field applying part operable from outside of thenarrow pipe and pass through a water-insoluble gel substance layer.

LITERATURE OF RELATED ART Patent Literature

[Patent literature 1] WO 2012/086243

Non-Patent Literature

-   [Non-patent literature 1] Clinical Chemistry 51: 882-890, 2005, Mar.    3, 2005-   [Non-patent literature 2] “FDA Issues Another Emergency Use    Authorization for Commercial H1N1 Flu Test to Quest Diagnostics'    Focus Diagnostics”, Focus Diagnostics Co., Ltd, Oct. 17, 2009-   [Non-patent literature 3] “GC series Magtration Genomic DNA Whole    Blood”, Precision System Science Co., Ltd, December, 2008

SUMMARY

In the operation pipe disclosed in patent literature 1, a samplecontaining magnetic particles is introduced from an open end of a pipeconstituting the operation pipe. The sample is obtained by crushing ordissolving biological samples such as blood or cells, and biologicalcomponents such as nucleic acids is adsorbed to the magnetic particles.The magnetic particles adsorbing the biological components can becollected by an external magnetic field. Furthermore, the magneticparticles adsorbing the biological components can be moved in the pipeby movement of the external magnetic field. Inside the pipe, a cleaningliquid layer for washing away contaminants contained in the biologicalsample and an elution liquid layer for liberating the biologicalcomponents such as nucleic acids are filled as liquid reagent layers,and the cleaning liquid layer and the elution liquid layer arepartitioned by a water-insoluble gel layer so as not to be mixed. Whenthe external magnetic field is gently moved, the magnetic particlesadsorbing the biological components such as nucleic acids can follow themovement and pass through the gel layer without mixing the cleaningliquid and the elution liquid. On the other hand, when the externalmagnetic field is rapidly reciprocated, the magnetic particles in theliquid reagent layer cannot follow the movement of the external magneticfield and are dispersed in the liquid. By this operation, thecontaminant components contained in the biological sample are washedaway in the cleaning liquid layer, and the biological components such asnucleic acids are liberated from the magnetic particles in the elutionliquid layer.

The cleaning liquid layer, the eluent liquid layer, and thewater-insoluble gel layer filled in the pipe constituting the operationpipe disclosed in patent literature 1 are filled based on a distance inthe longitudinal direction of the pipe. The reason is that accuratesetting of a movement position can be made based on the distance in thesetting of a driving program for moving the external magnetic field. Onthe other hand, when an inner diameter of the pipe varies, a volume ofthe liquid reagent in the liquid reagent layer varies due to thevariation. In particular, when the liquid reagent is an elution liquidthat liberates biological components such as nucleic acids, variation inthe volume of the elution liquid causes variation in the concentrationof the biological components such as nucleic acids eluted in the elutionliquid. As a result, accurate recovery rate evaluation of the biologicalcomponents such as nucleic acids is hindered.

The disclosure provides an operation pipe and a device equipped with theoperation pipe, which enable accurate recovery rate evaluation ofbiological components such as nucleic acids recovered in an elutionliquid.

That is, the disclosure includes the following aspects.

[1]An operation pipe for operating target components, including: a hollowpipe, having a closable open end for supplying a sample containing thetarget components on one side and a closed end on the other side, andhaving an operation pipe portion a on the open end side and a recoverypipe portion b on the closed end side; an operation medium, which isfilled in the operation pipe portion a so that gel layers and aqueousliquid layers are alternately multi-layered in the longitudinaldirection of the hollow pipe, wherein a layer length of the gel layersand a layer length of the aqueous liquid layers are determined by thelength in the longitudinal direction of the hollow pipe; a recoverymedium, which is filled in the recovery pipe portion b so that a gellayer and an aqueous liquid layer which is in contact with the closedend are multi-layered, wherein the aqueous liquid layer in contact withthe closed end has a predetermined volume, and the layer length of thegel layer is determined by the length in the longitudinal direction ofthe hollow pipe; and magnetic particles for capturing and transportingthe target components; wherein the magnetic particles pass through thegel layer in a gel state and move in the longitudinal direction of theoperation pipe due to application of a magnetic field.

The open end is preferably closed so that all or a part of the open endcan be opened and closed. In FIGS. 1B and 1C, an example of a preferableopen end, an example of the hollow pipe having the operation pipeportion a on the open end side and the recovery pipe portion b on theclosed end side, and an example of the operation pipe are shown.

[2]

The operation pipe according to [1], wherein an inner diameter of thehollow pipe is 0.1 mm-5 mm.[3]

The operation pipe according to [1] or [2], wherein a volume of theaqueous liquid layer in contact with the closed end is 1 μL-1000 μL.

[4]

The operation pipe according to any one of [1] to [3], wherein theoperation pipe portion a and the recovery pipe portion b are separable.

[5]

The operation pipe according to any one of [1] to [4], wherein thematerial of the hollow pipe is selected from a group consisting ofpolyethylene, polypropylene, fluororesin, polyvinyl chloride,polystyrene, polycarbonate, acrylonitrile-butadiene-styrene copolymer,acrylonitrile-styrene copolymer, acrylic resin, polyvinyl acetate,polyethylene terephthalate, cyclic polyolefin, and glass.

[6]

The operation pipe according to any one of [1] to [5], wherein an innerdiameter of the open end is larger than an inner diameter of theoperation pipe portion a and an inner diameter of the recovery pipeportion b.

[7]

The operation pipe according to any one of [1] to [6], wherein thehollow pipe has optical transparency.

[8]

The operation pipe according to any one of [1] to [7], wherein surfaceroughness of an inner surface of the hollow pipe is 0.1 am or less.

[9]

The operation pipe according to any one of [1] to [8], wherein a lengthof the gel layer in the longitudinal direction of the hollow pipe is1-20 mm, the gel layer being filled in the operation pipe portion a andthe recovery pipe portion b.

[10]

The operation pipe according to any one of [1] to [9], wherein a lengthof the aqueous liquid layer in the longitudinal direction of the hollowpipe is 0.5-30 mm, the aqueous liquid layer being filled in theoperation pipe portion a.

[11]

The operation pipe according to any one of [1] to [10], wherein themagnetic particles are particles having a binding force or adsorptionforce to nucleic acids that are used as target components, the aqueousliquid layer in the operation medium is an aqueous liquid layercontaining a liquid that liberates nucleic acids and binds or adsorbsthe nucleic acids to the magnetic particles and/or an aqueous liquidlayer containing a cleaning liquid of the magnetic particles, and theaqueous liquid layer in the recovery medium which is in contact with theclosed end is an aqueous liquid layer containing a liquid that liberatesnucleic acids.

[12]

The operation pipe according to [11], wherein the aqueous liquid layerin the recovery medium which is in contact with the closed end is anaqueous liquid layer further containing a reverse transcription reactionliquid and/or a nucleic acid amplification reaction liquid.

[13]

The operation pipe according to [12], wherein the aqueous liquid layerin the recovery medium which is in contact with the closed end is anaqueous liquid layer further containing a fluorescent dye that is usedto be specifically bound to the target components and detect the targetcomponents by generating fluorescence by light irradiation.

[14]

A device, including: the operation pipe according to [13]; a magneticfield applying part, which is capable of moving the magnetic particlesin the longitudinal direction of the operation pipe by applying amagnetic field to the operation pipe; and an optical detection part,which irradiates light to the recovery pipe portion b and detectsfluorescence generated from the fluorescent dye specifically bound tothe target components.

[15]

A device including a plurality of operation pipes according to any oneof [1] to [13], and further including a magnetic field applying partcapable of simultaneously moving, for the plurality of operation pipes,the magnetic particles in the longitudinal direction of the operationpipe by simultaneously applying a magnetic field to the plurality ofoperation pipes.

[16]

The device according to [15], wherein the magnetic field applying partincludes: a movable substrate capable of moving in the longitudinaldirection of the operation pipe; a magnetic field moving mechanism,which controls movement of the movable substrate toward the longitudinaldirection of the operation pipe; and a plurality of magnetic sources,which corresponds to the plurality of operation pipes and is held in themovable substrate.

[17]

A device including the operation pipe according to any one of [1] to[13], and a magnetic field applying part capable of moving the magneticparticles in the longitudinal direction of the operation pipe byapplying a magnetic field to the operation pipe, wherein the magneticfield applying part causes the magnetic field to perform amplitudemovement in the longitudinal direction of the operation pipe or causesthe magnetic field to perform rotational motion.

Effect

According to the disclosure, the volume of the elution liquid thatliberates biological components such as nucleic acids is constant, theelution liquid being filled in the recovery pipe portion b in a hollowpipe constituting an operation pipe and constituting an aqueous liquidlayer in contact with a closed end of the hollow pipe, and thus accuraterecovery rate evaluation of the biological components such as nucleicacids recovered in the elution liquid can be made. A gel layer and anaqueous liquid layer other than the aqueous liquid layer in contact withthe closed end of the hollow pipe, which is the elution liquid forliberating the biological components such as nucleic acids, are filledwith a thickness determined by the length in the longitudinal directionof the hollow pipe, and thus it is unnecessary to change or modify adrive program for moving an external magnetic field.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C are longitudinal-sectional views of examples of anoperation pipe of the disclosure. A and B described in FIG. 1Crespectively represent an operation portion A and a recovery portion B.The operation portion A includes an operation pipe portion acorresponding in a hollow pipe and an operation medium filled in thepipe portion a. The recovery portion B includes a recovery pipe portionb corresponding in the hollow pipe and a recovery medium filled in thepipe portion b.

FIGS. 2A to 2C show an example of a manufacturing method of theoperation pipe of the disclosure.

FIGS. 3A to 3O show a process in which the operation pipe of thedisclosure shown in FIGS. 1A to 1C is used to extract and purify nucleicacids from a nucleic acid-containing sample.

FIGS. 4A to 4H show a process in which another example of the operationpipe of the disclosure is used to extract and purify nucleic acids fromthe nucleic acid-containing sample, and analysis performed by a reversetranscription reaction and a PCR reaction are further performed.

FIG. 5 is a perspective view showing an example of a device that enablesa simultaneous operation in the operation pipe by using a plurality ofoperation pipes of the disclosure to make multiple channels.

FIG. 6 is a cross-sectional view of a variation example of a magneticfield applying part (movable magnet plate) shown in FIG. 5.

FIG. 7A is a longitudinal-sectional view of the variation example of themagnetic field applying part (movable magnet plate) shown in FIG. 5,FIG. 7B is a longitudinal-sectional view of a variation example of aholding part (holding substrate), and FIG. 7C is alongitudinal-sectional view of a part including the operation pipe heldon the holding part.

FIG. 8 is a result obtained in Example 1 in which the process shown inFIGS. 3A to 3O is performed.

DESCRIPTION OF THE EMBODIMENTS

[1. Operation of Target Components]

[1-1. Target Components]

Target components operated in the disclosure is not particularly limitedas long as the target components can be operated in common aqueousliquids, emulsions, or hydrogels, and may be any in vivo components andnon-in vivo components. The in vivo components include biomolecules suchas nucleic acids (including DNA and RNA), proteins, lipids, and sugars.The non-in vivo components include non-biomolecules such as artificial(both chemical and biochemical) modifiers, labelled bodies, mutants andthe like of the biomolecules, non-biomolecules derived from naturalproducts, and other components that can be operated in an aqueoussystem.

The target components can usually be provided in the form of a samplecontaining the target components. The sample include, for example,biological samples such as animal and plant tissues, body fluids andexcreta, and biomolecule-containing bodies such as cells, protozoa,fungi, bacteria and viruses. The body fluids include blood, sputum,cerebrospinal fluid, saliva, and milk and may be combinations thereof;and the excreta include feces, urine, and sweat and may be combinationsthereof. The cells include leukocytes and platelets in blood orexfoliated cells of oral cells and other mucosal cells and may becombinations thereof. These samples can also be obtained as clinicalswabs. In addition, the above sample can also be prepared, for example,in the form of a liquid mixture of a cell suspension, a homogenate, anda cell lysate. In addition, the sample containing the target componentscan also be obtained by applying treatments such as modification,labelling, fragmentation, and mutation to the above sample.

The sample containing the target components may also be prepared inadvance by subjecting the above sample to pre-treatment. Thepre-treatment includes, for example, a treatment for performingextraction, separation, and purification of the target components or thetarget component-containing body from the sample containing the targetcomponents, and the like. However, since this pre-treatment can beperformed in the operation pipe of the disclosure, it is not alwaysnecessary to perform the pre-treatment before the sample is added intothe operation pipe. By performing the pre-treatment in the operationpipe of the disclosure, a contamination problem that is a concern in thepre-treatment of the sample can be avoided.

[1-2. Operation]

[1-2-1. Operation Form]

In the disclosure, the sample containing the target components is addedto the operation pipe illustrated as 1 in FIGS. 1A to 1C, and the targetcomponents are operated in the operation pipe. Operations of the targetcomponents in the disclosure include supplying the target components tovarious treatments and transporting the target components among aplurality of environments for performing the various treatments. Theoperation pipe is filled with gel layers and aqueous liquid layers. Forexample, in the form illustrated in FIGS. 1A to 1C, the layersrepresented by 2 g and 3 g consist of gels (gel plugs), and the layerrepresented by 31 consists of an aqueous liquid. The layer representedby 4 is an aqueous liquid layer in contact with the closed end of ahollow pipe constituting the operation pipe of the disclosure, and mayalso be a hydrogel as long as the aqueous liquid can maintain a gelstate. The aqueous liquid and the hydrogel construct an environment forperforming a treatment of the target components. Accordingly, morespecifically, the operations of the target components in the disclosureinclude supplying the target components to treatments in the aqueousliquid or hydrogel and transporting the target components among aplurality of environments for performing the treatments via a gel plug.

[1-2-2. Treatment of Target Components]

The treatments to which the target components are supplied include thetreatment accompanied by a substance change of the target components andthe treatment accompanied by a physical change of the target components.

The treatment accompanied by a substance change of the target componentsmay be any treatment as long as a different substance is newly generatedby generating or breaking a bond between substrates. More specifically,a chemical reaction and a biochemical reaction are included. Thechemical reaction may be any reaction accompanied by compounding,decomposition, oxidation and reduction. In the disclosure, generally,treatments that are performed in an aqueous liquid are included. Thebiochemical reaction may be any reaction accompanied by a change ofbiological substances, and usually refers to an in vitro reaction. Forexample, reactions based on a synthesis system, a metabolic system andan immune system of biological substances such as nucleic acids,proteins, lipids, sugars and the like are included.

The treatment accompanied by a physical change of the target componentsmay be any treatment not accompanied by the above substance change. Morespecifically, denaturation (for example, when the target components arebiopolymers or other polymers containing nucleic acids or proteins),dissolution, mixing, emulsification, dilution, and the like of thetarget components are included.

Accordingly, operations such as separation, extraction, purification,elution, recovery, and analysis of the target components can beperformed by the treatment in the disclosure. By these operations,isolation, detection, identification and the like of the targetcomponents can be finally performed.

The treatments in the disclosure include not only the desired treatments(treatments in a process in which effects of isolation, detection,identification and the like of the target components are directlyobtained), but also a pre-treatment and/or a post-treatment associatedtherewith as necessary. For example, when the target components arenucleic acids, a nucleic acid amplification reaction or a nucleic acidamplification reaction and analysis of amplification products areperformed, but extraction (cell lysis) and/or purification (cleaning) ofthe nucleic acids from a nucleic acid-containing sample and the like areessential as the pre-treatments thereof. In addition, recovery and thelike of the amplification products may be performed as thepost-treatments.

[1-2-3. Transport of Target Components]

The transport of the target components is performed by magneticparticles and a magnetic field applying part. The magnetic particles arepresent in the operation pipe during operation, and can transport thetarget components by moving the target components in the operation pipein a state that the target components are captured by being bound andabsorbed to the surface of the magnetic particles. The magneticparticles can be dispersed in the aqueous liquid layer in the operationpipe, and are usually aggregated in the aqueous liquid layer due togeneration of a magnetic field from outside of the operation pipe by themagnetic field applying part. The aggregated magnetic particles can movealong with changes of the magnetic field that is generated from outsideof the operation pipe by the magnetic field applying part. Theaggregated magnetic particles can move in the gel layer. By utilizingthe thixotropic property (thixotropic property) of the gel described in3-2-3, the aggregated magnetic particles can pass through the gel layerwithout destroying the gel layer. In the gel, the aggregated magneticparticles are accompanied by the target components by binding oradsorption. Strictly speaking, the group of the aggregated magneticparticles is coated with a very small amount of aqueous liquid.Accordingly, components other than the target components may beaccompanied. However, since the amount of the coated aqueous liquid isvery small, almost no aqueous liquid is contained. Therefore, thetransport of the target components can be performed very efficiently.

[2. Operation Pipe]

[2-1. Structure of Operation Pipe]

The structure of the operation pipe of the disclosure is described withreference to FIGS. 1A-1C (in the following description, the verticaldirection uses FIGS. 1A-1C as a reference). The hollow pipe constitutingthe operation pipe has an upper end opened for sample feeding, and theopen end is preferably closable from the viewpoint of contamination. Alower end of the hollow pipe is a closed end. Usually, the hollow pipeconstituting the operation pipe has a substantially circular crosssection, but pipes having other shapes of cross section are notexcluded. The pipe is filled with an operation medium in which aqueousliquid layers 1 and gel layers g are alternately multi-layered in thelongitudinal direction of the pipe. FIGS. 1A-1C illustrate three aspectsFIGS. 1A-1C in which forms of an upper portion and a lower portion ofthe operation pipe are different. However, the upper portion and thelower portion can be arbitrarily combined and are not limited to thecombinations shown in FIGS. 1A-1C.

The upper open end of the pipe is a sample supply portion 5 forsupplying the sample containing the target components, and the samplesupply portion 5 that is an open end may be temporarily opened (see FIG.1A), or all or a part of the sample supply portion 5 may be openablyclosed (shown in FIG. 1B). By using a septum having a check valvefunction as an example that a part is openably closed, it is possible tosupply a sample by puncturing with an injection needle that cansubstantially maintain a sealed state (shown in FIG. 1C). It ispreferable to close the sample supply portion 5 that is an open endbecause a completely sealed system can be constructed. By constructing acompletely closed system, contamination from outside during operationcan be prevented, and thus the operation pipe is very effective. Theinner diameter of the sample supply portion 5 may be the same as aninner diameter of the pipe portion a filled with the gel layer and theaqueous liquid layer which are operation mediums (shown in FIG. 1A), ormay be formed to have a wider inner diameter from the viewpoint ofoperability during supply of the sample (shown in FIGS. 1B and 1C).

In the aspects illustrated in FIGS. 1A and 1B, the pipe is integrallyformed. In the aspect illustrated in FIG. 1C, the pipe is configured bythe operation pipe portion a and the recovery pipe portion b. The upperend and the lower end of the operation pipe portion a are open. Theupper end of the recovery pipe portion b is open and the lower end isclosed. In the operation pipe portion a and the recovery pipe portion b,one end of the pipe portion a and the open end of the pipe portion b areconnected. The operation pipe portion a and the recovery pipe portion bmay have a separable shape, or may have a shape not consideringseparation (a shape that cannot be separated).

The operation pipe portion a is filled with a gel layer 2 g that closesone end and a multi-layer that is multi-layered on the gel layer 2 g,that is, an operation medium 3. The operation medium 3 is configured sothat aqueous liquid layers 31 and gel layers 3 g are alternatelymulti-layered. The part configured by the operation pipe portion a andthe operation medium that is the filling of the operation pipe portion ais described as an operation portion A. The recovery pipe portion b isfilled so that a gel layer in contact with the operation pipe portion aand an aqueous liquid layer in contact with the closed end at the lowerend are multi-layered. The aqueous liquid layer in contact with theclosed end is represented by a recovery medium 4. The part configured bythe recovery pipe portion b, the recovery medium 4 that is the fillingof the recovery pipe portion b, and the gel layer in contact with theoperation pipe portion a is described as a recovery portion B. Theoperation portion A and the recovery portion B may be provided in aconnected state or may be provided in an independent state. In therecovery portion B, the gel layer in contact with the operation pipeportion a has a function of preventing the recovery medium 4 fromflowing out in a state that the recovery portion B is separated orindependent. The recovery portion B may be configured by a recovery pipeportion b that is not filled with the gel layer and is filled only withthe aqueous liquid layer.

[2-2. Size of Operation Pipe]

The approximate inner diameter of the hollow pipe constituting theoperation pipe is, for example, 0.1 mm-5 mm, preferably 1-2 mm. If theapproximate inner diameter is in this range, the operation pipe can havegood operability. When the substantially inner diameter is below theabove range, a pipe wall becomes thick to maintain the strength of thehollow pipe; as a result, a distance between the magnetic particles andthe magnet increases, and a magnetic force reaching the magneticparticles becomes weak, which may cause operational problems. On theother hand, when the inner diameter of the hollow pipe exceeds the aboverange, an interface between the multi-layer of the gel layer and theaqueous liquid layer constituting the operation medium tends to beeasily disturbed due to an impact from outside or an influence ofgravity. Besides, in the disclosure, the pipe having an inner diameterof 0.1 mm or less is not excluded as long as the capillary material canwithstand high-precision processing. The length in the longitudinaldirection of the operation pipe is, for example, 1-30 cm, preferably5-15 cm.

Besides, as shown in FIGS. 1B and 1C, when the sample supply portion 5is formed in a manner that the inner diameter is wider, the approximateinner diameter of the sample supply portion 5 exceeds the above rangeand may be 10 mm or less, preferably 5 mm or less. It is preferable fromthe viewpoint of workability during sample supply that the sample supplyportion has a wider inner diameter. When the wider inner diameterexceeds the above range, for example, when a plurality of operationpipes are processed at the same time, the operation pipes come intocontact with each other, and integration of the device tends todecrease.

[2-3. Material of Pipe]

The material of the hollow pipe constituting the operation pipe is notparticularly limited. For example, in order to reduce movementresistance when the target components and a small amount of liquid movetogether with the magnetic particles in the gel layer, the inner wallwhich is a conveyance surface is smooth and water-repellent. Thematerial that gives such properties includes resin materials such aspolyethylene, polypropylene, fluororesin (Teflon (registeredtrademark)), polyvinyl chloride, polystyrene, polycarbonate,acrylonitrile-butadiene-styrene copolymer (ABS resin),acrylonitrile-styrene copolymer (AS resin), acrylic resin, polyvinylacetate, polyethylene terephthalate, cyclic polyolefin and the like. Theresin materials are preferable in terms that the layer in the operationpipe is unlikely to be disturbed even if the operation pipe is droppedor bent and is highly robust. The material of the pipe may be glass ifnecessary for transparency, heat resistance and/or workability. Thematerial of the sample supply portion 5, the operation pipe portion a,and the recovery pipe portion b may be the same or be different.

[2-4. Physical Properties of Hollow Pipe]

From the viewpoint of visibility during operation and from the viewpointof optical detection in a case of measurement of absorbance,fluorescence, chemiluminescence, bioluminescence, refractive indexchange and the like from outside of the pipe, the material of the hollowpipe preferably has optical transparency.

The conveyance surface constituting the inner wall of the pipe ispreferably a smooth surface in order to move a small amount of liquidmass containing the target components together with the magneticparticles in the gel layer; particularly, the surface roughness ispreferably Ra=0.1 μm or less. For example, when a permanent magnet isbrought closer to the pipe from outside and the small amount of liquidmass containing the target components moves due to the changes of themagnetic field, the magnetic particles move while being pressed againstthe conveyance surface, but by the conveyance surface having surfaceroughness of Ra=0.1 μm or less, followability of the magnetic particlesto the changed magnetic field can be sufficiently provided.

[3. Filling in Operation Pipe]

[3-1. Operation Medium and Recovery Medium]

The operation pipe is at least filled with, as the operation medium, themulti-layers in which the aqueous liquid layers and the gel layers arealternately multi-layered. The uppermost layer may be a gel layer (FIG.1A) or an aqueous liquid layer (FIGS. 1B and 1C). When the uppermostlayer is an aqueous liquid layer, the layer may contain magneticparticles 6 (FIG. 1C) or may not contain the magnetic particles 6 (FIGS.1A and 1B). The lowermost layer may be a gel layer or an aqueous liquidlayer (FIGS. 1A-1C).

As shown in FIGS. 1A and 1B, when the hollow pipe constituting theoperation pipe is integrally formed, all layers filled in the pipe maybe in contact with each other. As shown in FIG. 1C, when the hollow pipeconstituting the operation pipe consists of the operation pipe portion aand the recovery pipe portion b, the recovery pipe portion b may befilled with an aqueous liquid only as the recovery medium or may also befilled with a gel layer on the aqueous liquid layer. The gel layer 2 gfilled at the lowermost end of the operation pipe portion a and theaqueous liquid layer or gel layer filled at the uppermost end of therecovery pipe portion b may be in contact with each other, or may not bein contact with a layer of gas interposed therebetween (FIG. 1C).

The number and order of the layers filled in the hollow pipe are notparticularly limited, and can be appropriately determined by thoseskilled in the art based on the number and order of the operationprocesses for supplying the target components. Each of the plurality ofaqueous liquid layers filled in one hollow pipe preferably consists oftwo or more different types of aqueous liquids. As the aqueous liquidconstituting each layer, liquids that construct environment necessaryfor each of a treatment process and a reaction process for supplying thetarget components can be used in order from the upper end side of thehollow pipe. Each of the plurality of gel layers filled in one hollowpipe may consist of different types of gels or may consist of the sametype of gel. For example, when a heating treatment or reaction isperformed in a part of the plurality of aqueous liquid layers, a gelhaving a high sol-gel transition point for which a gel state or agel-sol intermediate state can be maintained even at a temperaturenecessary for the heating can be used in the gel layer adjacent to theaqueous liquid layer only, and a gel having a relatively low sol-geltransition point can be used in other gel layers. In addition, anyoneskilled in the art can appropriately select a gel having propercharacteristics corresponding to the characteristics or volume of theaqueous liquid constituting adjacent aqueous liquid layers.

The gel layer serves as a plug (gel plug) that partitions the aqueousliquid layer on both sides in the longitudinal direction of the hollowpipe in the operation pipe. As for the layer length, those skilled inthe art can appropriately determine the length of the layer thatfunctions as a plug in consideration of the inner diameter and thelength of the pipe, the amount of the magnetic particles conveyed by themagnetic field applying part and the like. For example, the layer lengthmay be 1-20 mm, preferably 3-10 mm. When the layer length is below therange, the gel layer tends to lack intensity as a plug. When the layerlength is above the range, the operation pipe becomes long, andoperability, durability and fillability of the operation pipe tend todeteriorate.

The aqueous liquid layer filled in the operation pipe portion a providesan environment of treatment, reaction or the like in which the samplecontaining the target components is supplied. As for the layer length ofthe aqueous liquid layer, those skilled in the art can appropriatelydetermine the layer length that gives an aqueous liquid amount forachieving a desired treatment or reaction for the target components, inconsideration of the inner diameter or length of the hollow pipe, theamount of the target components, the type of the treatment or reactionin which the target components are supplied and the like. For example,the layer length is 0.5-30 mm, preferably 3-10 mm. When the layer lengthis below the range, the treatment or reaction for the target componentsmay not be sufficiently achieved, and the plug may be droplet-like andthe magnetic particles may not be able to bind to the reagents. When thelayer length is above the range, the aqueous liquid layer is oftenrelatively much thicker compared with the gel layer, which may cause thesame problem as the gel plug, and the interface of the multi-layer tendsto be easily disturbed when the specific gravity of the aqueous liquidis larger than that of the gel.

The aqueous liquid layer filled in the recovery pipe portion b is anelution liquid of the target components, and provides an environment forliberating the target components from the magnetic particles. Theaqueous liquid layer is filled in the hollow pipe so as to have apredetermined volume. Accordingly, the layer length also varies withvariations in the inner diameter or length of the hollow pipe. Thevolume of the aqueous liquid layer can be appropriately determined bythose skilled in the art in consideration of the amount of the targetcomponents, the type of the treatment or reaction in which the targetcomponents are supplied and the like so that the recovery rate of thetarget components recovered in the elution liquid can be accuratelyevaluated. For example, the volume of the aqueous liquid layer is 1μL-1000 μL, preferably 50 μL-300 μL.

When the gel layer consists of a hydrogel, the hydrogel layer can notonly function to partition the reagents but also provide an environmentof the treatment or reaction or the like in which a sample containingthe target components is supplied in the same manner as the aqueousliquid layer. In this case, the hydrogel layer may also be longer thanthe aqueous liquid layer.

[3-2. Type of Gel]

The gel layer consists of a chemically inert substance that is insolubleor poorly soluble in a liquid constituting the aqueous liquid layer whenmulti-layered with the aqueous liquid in the hollow pipe. Beinginsoluble or poor-soluble in liquid means that the solubility in theliquid at 25° C. is approximately 100 ppm or less. The chemically inertsubstance refers to substance that has no chemical effect on the targetcomponents and the aqueous liquid or the hydrogel during operation ofthe target components (that is, the treatment of the target componentsin the aqueous liquid or the hydrogel and transport of the targetcomponents via the gel plug). The gel in the disclosure includes bothorganogel and hydrogel.

[3-2-1. Organogel Gel]

Usually, the organogel can be obtained by adding a gelling agent to awater-insoluble or poorly water-soluble liquid substance for gelation.

[3-2-1-1. Water-Insoluble or Poorly Water-Soluble Liquid Substance]

As the water-insoluble or poorly water-soluble liquid substance, oilthat has a solubility in water at 25° C. of approximately 100 ppm orless and that is liquid-like at room temperature (20° C.±15° C.) can beused. For example, one or a combination of two or more from a groupconsisting of liquid oil, ester oil, hydrocarbon oil, and silicone oilcan be used.

The liquid oil includes linseed oil, camellia oil, mackerel demia nutoil, corn oil, mink oil, olive oil, avocado oil, southern power oil,castor oil, safflower oil, kyounin oil, cinnamon oil, jojoba oil, grapeoil, sunflower oil, almond oil, rapeseed oil, sesame oil, wheat germoil, rice germ oil, rice bran oil, cottonseed oil, soybean oil, peanutoil, tea seed oil, evening primrose oil, egg yolk oil, liver oil, palmoil, palm oil, palm nuclear oil, and the like.

The ester oil includes octanoic esters such as cetyl octanoate, lauricesters such as hexyl laurate, myristate esters such as isopropylmyristate and octyldodecyl myristate, palmitate esters such as octylpalmitate, stearic acid esters such as isocetyl stearate, isostearicacid esters such as isopropyl isostearate, isopalmitic acid esters suchas octyl isopalmitate, oleic acid esters such as isodecyl oleate, adipicacid esters such as isopropyl adipate, sebacic acid esters such as ethylsebacate, malate esters such as isostearyl malate, glycerintrioctanoate, glycerin triisopalmitate, and the like.

The hydrocarbon oil includes pentadecane, hexadecane, octadecane,mineral oil, liquid paraffin, and the like. The silicone oil includesdimethylpolysiloxane, methylphenylpolysiloxane and other phenylgroup-containing silicone oil, methylhydrogenpolysiloxane, and the like.

[3-2-1-2. Gelling Agent]

As the gelling agent, an oil gelling agent selected from a groupconsisting of hydroxy fatty acid, dextrin fatty acid ester, and glycerinfatty acid ester can be used alone or in combination of two or more.

The hydroxy fatty acid is not particularly limited as long as it is afatty acid having a hydroxyl group. Specifically, the hydroxy fatty acidincludes, for example, hydroxymyristic acid, hydroxypalmitic acid,dihydroxypalmitic acid, hydroxystearic acid, dihydroxystearic acid,hydroxymargaric acid, ricinoleic acid, ricinaleic acid, linolenic acid,and the like. Among these, hydroxystearic acid, dihydroxystearic acid,and ricinoleic acid are particularly preferable. These hydroxy fattyacids may be used alone or in combination of two or more. In addition,an animal and vegetable oil fatty acid (for example, castor oil fattyacid, hydrogenated castor oil fatty acid, and the like) that is amixture of these hydroxy fatty acids can also be used as the hydroxyfatty acid.

The dextrin fatty acid esters include, for example, dextrin myristate(trade name “Leopard MKL”, manufactured by Chiba Flour Milling Co.,Ltd.), dextrin palmitate (trade names “Leopard KL”, “Leopard TL”, bothmanufactured by Chiba Flour Milling Co., Ltd.), dextrin(palmitate/2-ethylhexanoate) (trade name “Leopard TT”, manufactured byChiba Flour Milling Co., Ltd.), and the like.

The glycerin fatty acid esters include glyceryl behenate, glyceryloctastearate, glyceryl eicoate, and the like, and one or more of theseglycerin fatty acid esters may be used in combination. Specifically, theglycerin fatty acid esters can include trade name “TAISET 26”(manufactured by Taiyo Chemical Co., Ltd.) containing 20% of glycerylbehenate, 20% of glyceryl octastearate and 60% of hydrogenated palm oil,trade name “TAISET 50” (manufactured by Taiyo Kagaku Co., Ltd.)containing 50% of glyceryl behenate and 50% of glyceryl octastearate,and the like.

The gelling agent can be used, of which the content added to thewater-insoluble or poorly water-soluble liquid substance is equivalentto, for example, 0.1-0.5 weight %, 0.5-2 weight %, or 1-5 weight % ofthe total weight of the liquid substance. However, the gelling agent isnot limited hereto, and those skilled in the art can appropriatelydetermine the amount to a degree at which the desired gel and sol statecan be achieved.

The gelling method can be appropriately determined by those skilled inthe art. Specifically, a water-insoluble or poorly water-soluble liquidsubstance is heated, a gelling agent is added to the heated liquidsubstance, the gelling agent is completely dissolved and then cooled,and thereby the liquid substance can be gelled. A heating temperaturemay be determined in consideration of the physical properties of theliquid substance and the gelling agent that are used. For example, theheating temperature may be preferably about 60-70° C. The gelling agentis dissolved for the liquid substance in a heated state; at this time,it is preferable that the gelling agent is dissolved while being gentlymixed with the liquid substance. Cooling is preferably performed slowly.For example, the cooling can be performed over a period of about onehour to two hours. For example, the cooling can be completed when thetemperature is lowered to room temperature (20° C.±15° C.) or less,preferably 4° C. An aspect to which a preferable aspect of the gellingmethod is applied includes, for example, an aspect in which theabove-described TAISET 26 (manufactured by Taiyo Kagaku Co., Ltd.) isused.

[3-2-2. Hydrogel]

As the hydrogel, for example, the hydrogel prepared by equilibrating andswelling hydrogel materials in water or an aqueous liquid can be used,the hydrogel materials including gelatin, collagen, starch, pectin,hyaluronic acid, chitin, chitosan or alginic acid and derivativesthereof. Among the hydrogels, it is preferable to use a hydrogelprepared from gelatin. In addition, the hydrogel can also be obtained bychemically cross-linking the above hydrogel materials or processing theabove hydrogel materials with the gelling agents (for example, salts ofalkaline metals/alkaline earth metal such as lithium, potassium andmagnesium, or salts of transition metal such as titanium, gold, silverand platinum, and silica, carbon, alumina compound or the like). Thesechemical cross-linking and gelling agents can be easily selected bythose skilled in the art.

In particular, when the hydrogel provides an environment of treatment orreaction or the like in which a sample containing the target componentsis provided in the same manner as the aqueous liquid, the hydrogel isappropriately prepared by those skilled in the art so as to have acomposition suitable for the treatment or reaction. The hydrogelincludes, for example, a DNA hydrogel (P-gel) based onpolydimethylsiloxane capable of synthesizing proteins. This hydrogel isconfigured by DNA which is used as a part of gel scaffold. When thetarget component is a substrate for protein synthesis, this hydrogel canbe supplied to a reaction for obtaining a protein from the targetcomponent (the more specific aspect can be appropriately determined bythose skilled in the art with reference to Nature Materials 8, 43 2-437(2009), and Nature Protocols 4: 1759-1770 (2009)). The produced proteincan be recovered, for example, by using the magnetic particles having anantibody specific for the protein.

[3-2-3. Gel Characteristics]

The gel filled in the hollow pipe has a characteristic of causing thesol-gel transition at a certain temperature. The sol-gel transitionpoint may be in a range of 25-70° C. The generation of the sol-geltransition point in this range is desirable in a reaction system thatrequires fluidity obtained by solification in recovery or the like. Thesol-gel transition point varies depending on conditions such as the typeof organogel material (oil) or hydrogel material, the type of gellingagent, the added amount of gelling agent, and the like. Accordingly,each condition is appropriately selected by those skilled in the art soas to obtain a desired sol-gel transition point.

The gel plug can be fixed in a predetermined position in the pipe byclamping the aqueous liquid in the hollow pipe from both sides in thelongitudinal direction of the pipe. On the other hand, the magneticparticles can also be moved even in the gel by a magnetic fieldoperation from outside, and can pass through the gel as a result. Thereason is the thixotropic property of the gel (thixotropy). That is, themagnetic particles inside the pipe give a shearing force to the gelalong the conveying surface due to the magnet movement outside the pipe,and the gel in the forward direction of the magnetic particles solatesand fluidizes, and thus the magnetic particles proceed directly.Moreover, the sol released from the shearing force after the passage ofthe magnetic particles returns quickly to the gel state, and thus nothrough hole caused by the passage of the magnetic particles is formedin the gel. By utilizing this phenomenon, the object can easily moveusing the magnetic particles as a transporter, and thus various chemicalenvironments in which the object is supplied can be switched in a veryshort time. For example, if the disclosure is used in a systemconsisting of a plurality of chemical reactions using a plurality ofreagents, the treatment time of the object can be greatly shortened. Ifthe property of gelation at a temperature below room temperature isutilized, even a reagent that exhibits a liquid state at thattemperature can also be immobilized by being sandwiched by the gel plugsin the pipe. Therefore, the state in which the narrow pipe is filledwith the liquid reagents in advance can be maintained from the time ofdevice manufacture until the delivery to the user, and the liquidreagents can be stably supplied. Furthermore, reagent collecting anddispensing operations for each work process are not necessary, labourreduction and time saving can be achieved, and deterioration of analysisaccuracy due to contamination can be prevented.

-   -   As for the physical properties of the gel, storage modulus E′ of        the dynamic viscoelasticity is preferably 10-100 kPa, more        preferably 20-50 kPa at room temperature (20° C.±15° C.). When        the storage modulus is below the range, the gel tends to lack        the intensity as a gel plug. When the storage modulus is above        the range, even magnetic particles having a particle size of        about several micrometres tend to be easily hindered in        movement. In the sol state, a kinematic viscosity may be 5        mm²/s-100 mm²/s, preferably 5 mm²/s-50 mm²/s, for example, about        20 mm²/s (50° C.).

[3-3. Type of Aqueous Liquid]

The aqueous liquid in the disclosure may be an aqueous liquid that isinsoluble or poorly soluble in the gel, and may be provided in the formof water, an aqueous solution or a creamy mixture of liquids calledemulsion, or a suspension in which fine particles are dispersed. Thecomponents of the aqueous liquid include all components that provide theenvironment of reaction and treatment in which the target components inthe disclosure are supplied.

More specific examples include a liquid for liberating components to beoperated in the disclosure into the aqueous liquid layer and binding oradsorbing the components to the surfaces of magnetic particles (that is,a liquid having an action of separating the target components fromcontaminants and promoting binding or adsorption to the surfaces ofmagnetic beads), a cleaning liquid for removing the contaminantscoexisting with the target components, an elution liquid for separatingthe target components adsorbed on the magnetic particles from themagnetic particles, a reaction liquid for constructing a reaction systemin which the target components are supplied, and the like. For example,when the target components are nucleic acids, the aqueous liquidincludes a reagent solution (cell lysate) for destroying cells andliberating the nucleic acids, and adsorbing the nucleic acids on thesilica-coated surfaces of the magnetic particles, a cleaning liquid forcleaning the magnetic particles and removing components other than thenucleic acids, an elution liquid (nucleic acid elution liquid) forseparating the nucleic acids from the magnetic particles, a nucleic acidamplification reaction liquid for performing nucleic acid amplificationreaction, and the like. Hereinafter, a case in which the targetcomponents are nucleic acids is illustrated, and the treatment liquidand reaction liquid for the nucleic acids and the treatment and reactionin which the nucleic acids are supplied are further described.

[3-3-1. Cell Lysate]

The cell lysate includes a buffer that contains chaotropic substances.The buffer can further include EDTA and any other chelating agent orTritonX-100 and any other surfactant. The buffer is based on, forexample, Tris-HCl and any other buffer. The chaotropic substanceincludes guanidine hydrochloride, guanidine isothiocyanate, potassiumiodide, urea and the like.

The chaotropic substance is a powerful protein denaturant, and has anaction of pulling proteins such as histones bound to the nucleic acidsaway from the nucleic acids and promoting adsorption on thesilica-coated surfaces of magnetic particles. The buffer agent can beused as an auxiliary agent that adjusts a pH environment in which thenucleic acids are easily adsorbed on the surfaces of the magneticparticles. The chaotropic substance also has an action of cell lysis(that is, an action of destroying cell membranes). However, in theaction of cell lysis, a surfactant contributes more than the chaotropicsubstance. A chelating agent can be used as an auxiliary agent thatpromote the cell lysis.

A specific protocol for extracting nucleic acids from a samplecontaining nucleic acids can be appropriately determined by thoseskilled in the art. In the disclosure, since the magnetic particles areused for transporting the nucleic acids in a droplet encapsulatingmedium, it is preferable to adopt a method using the magnetic particlesas the nucleic acid extraction method. For example, with reference toJapanese Patent Laid-Open 2-289596, a method for using magneticparticles to extract and purify nucleic acids from a sample containingnucleic acids can be implemented.

[3-3-2. Cleaning Liquid]

The cleaning liquid is preferably a solution capable of dissolvingcomponents other than the nucleic acids contained in the nucleicacid-containing sample (for example, proteins and sugars) or thereagents and other components used for other treatments performed inadvance such as nucleic acid extraction while the nucleic acids areadsorbed on the surfaces of the magnetic particles. Specifically, thecleaning liquid includes high salt concentration aqueous solutions suchas sodium chloride, potassium chloride and ammonium sulfate, alcoholaqueous solutions such as ethanol and isopropanol, and the like. Thecleaning of the nucleic acids is cleaning of the magnetic particles onwhich the nucleic acids are adsorbed. A specific protocol for thiscleaning can be appropriately determined by those skilled in the art. Inaddition, the number of times of the cleaning of the magnetic particleson which the nucleic acid are adsorbed can be appropriately selected bythose skilled in the art to a degree that undesired inhibition does notoccur during the nucleic acid amplification reaction. In addition, whenthe effect of inhibitory components can be ignored from the sameviewpoint, the cleaning process can also be omitted. The aqueous liquidlayer consisting of the cleaning liquid is prepared at least as manytimes as the number of times of the cleaning.

[3-3-3. Nucleic Acid Elution Liquid]

A buffer containing water, salt or the like can be used as the nucleicacid elution liquid. Specifically, a Tris buffer, a phosphate buffer,distilled water and the like can be used. A specific method forseparating the nucleic acids from the magnetic particles on which thenucleic acids are absorbed and eluting the nucleic acids into theelution liquid can also be determined appropriately by those skilled inthe art.

[3-3-4. Nucleic Acid Amplification Reaction Liquid]

In the nucleic acid amplification reaction liquid of the disclosure,various elements usually used in the nucleic acid amplification reactionat least include nucleic acids containing base sequences to be amplifiedand magnetic particles that adsorb the nucleic acids on the surfacesthereof.

Since the nucleic acid amplification reaction is not particularlylimited as described later, the various elements used in the nucleicacid amplification reaction can be appropriately determined by thoseskilled in the art based on the known nucleic acid amplification methodillustrated later and the like. Usually, the various elements includesalts such as MgCl₂, KCl, primers, deoxyribonucleotides, nucleic acidsynthases, and pH buffers. In addition, the above salts may beappropriately changed into other salts to use. In addition, substancesfor reducing non-specific priming, such as dimethyl sulfoxide, betaine,glycerol and the like, may be further added.

In addition to the above components, a blocking agent can be added tothe nucleic acid amplification reaction liquid in the disclosure. Theblocking agent can be used for the purpose of preventing deactivation ofDNA polymerase due to adsorption to the inner wall of a reaction vessel,the surfaces of the magnetic particles or the like. Specific examples ofblocking agents include bovine serum albumin (that is, BSA), otheralbumins, gelatin (that is, denatured collagen), proteins such as caseinand polylysine, peptides (both natural and synthetic), ficoll, polyvinylpyrrolidone, polyethylene glycol, and the like.

The nucleic acid amplification reaction of the disclosure is notparticularly limited, and for example, PCR method (U.S. Pat. No.4,683,195, No. 4683202, No. 4800159, and No. 4965188), LCR method (U.S.Pat. No. 5,494,810), Qβ method (U.S. Pat. No. 4,786,600), NASBA method(U.S. Pat. No. 5,409,818), LAMP method (U.S. Pat. No. 3,313,358), SDAmethod (U.S. Pat. No. 5,455,166), RCA method (U.S. Pat. No. 5,354,688),ICAN method (U.S. Pat. No. 3,433,929), TAS method (U.S. Pat. No.2,843,586), and the like can be used. In addition, a reversetranscription (RT) reaction can also be performed prior to the abovereaction. Those skilled in the art can appropriately select thecomposition of reaction liquid and the reaction temperature necessaryfor these nucleic acid amplification reactions.

Besides, when the nucleic acid amplification reaction is furtherperformed after the reverse transcription (RT) reaction, for example,when RT-PCR is performed, the RT reaction liquid layer can bemulti-layered on the PCR reaction liquid layer via the gel layer in therecovery portion B (for example, as illustrated in FIGS. 4A to 4H).

In a real-time nucleic acid amplification method, fluorescence detectioncan be performed on amplification products by a fluorescent dye capableof binding to a double-stranded DNA or by a probe labelled with thefluorescent dye. Detection methods in the real-time nucleic acidamplification method include the following methods.

For example, when it is possible to amplify a desired target only by ahighly specific primer, an intercalator method using SYBR (registeredtrademark) GREEN I or the like is used. An intercalator that emitsfluorescence by binding to a double-stranded DNA binds to thedouble-stranded DNA synthesized by the nucleic acid amplificationreaction, and emits fluorescence of a specific wavelength by irradiationof an excitation light. By detecting this fluorescence, the generationamount of the amplification products can be monitored. This method doesnot require design and synthesis of a fluorescently labelled probespecific to the target, and can be conveniently utilized in measurementof various targets.

In addition, when it is necessary to distinguish and detect similarsequences or when SNPs are typed, a fluorescently labelled probe methodis used. As an example, there is a TaqMan (registered trademark) probemethod for using an oligonucleotide in which 5′ terminal is modifiedwith a fluorescent substance and 3′ terminal is modified with a quenchersubstance as a probe. The TaqMan probe is specifically hybridized to atemplate DNA in an annealing step, but the quencher is present on theprobe and thus fluorescence emission is suppressed even when theexcitation light is irradiated. In an extension reaction step, when theTaqMan probe hybridized to the template is decomposed by 5′→3′exonuclease activity of the TaqDNA polymerase, the fluorescent dye isliberated from the probe, the suppression caused by the quencher isreleased and the fluorescence is emitted. By measuring the fluorescenceintensity, the generation amount of amplification products can bemonitored.

The principle of quantifying DNA in the real-time PCR by this method isdescribed below. First, a serially diluted standard sample of knownconcentration is used as the template to perform the PCR. Then, thenumber of cycles (threshold cycle; Ct value) reaching a certain amountof amplification products is obtained. A calibration curve is created byplotting the Ct value on the horizontal axis and an initial DNA amounton the vertical axis. For a sample of unknown concentration, the PCRreaction is also performed in the same conditions to obtain the Ctvalue. From this value and the above-described calibration curve, adesired DNA amount in the sample can be measured.

Furthermore, in an intercalator method, when the temperature of theliquid after the PCR reaction containing a fluorescent dye is graduallyincreased from 40° C. to about 95° C. and the fluorescence intensity iscontinuously monitored, a melting curve of the amplification productscan be obtained. The double-stranded DNA generated by the nucleic acidamplification reaction has a unique Tm value depending on the length ofDNA and the base sequence thereof. In other words, when the temperatureof droplets containing the DNA to which the fluorescent dye is bound isgradually increased, a temperature at which the fluorescence intensityrapidly decreases is observed. When the change amount of change influorescence intensity is examined, the temperature peak substantiallycoincides with the Tm value defined by the base sequence and the length.As a result, for example, data which is not the target gene and observedwhen a primer dimer occurs (that is, false positive data) can beexcluded from data regarded as positives. In a genetic testing,non-specific reactions also often occur due to contaminants in thesample, so it is important to eliminate the false positives.Accordingly, a determination can also be performed on whether thegenerated amplification products are unique to the target genes.

[3-3-5. Other Aqueous Liquids]

For any reaction and treatment other than the above reaction, thecomposition of each aqueous liquid can be easily determined by thoseskilled in the art. In addition, even when the target component is acomponent other than the above nucleic acid, the composition of eachaqueous liquid can be easily determined by those skilled in the art.

[4. Manufacturing Method of Operation Pipe]

As a manufacturing method of operation pipe, the following two methodsare described according to an aspect in which a hollow pipe to be filledwith multilayers being an operation medium is prepared.

[4-1. A Case in which One Hollow Pipe is Prepared for Manufacturing OneOperation Pipe]

The case in which this creation method is performed may be a case inwhich the hollow pipe is prepared in a state of being integrally formed,or a case in which the pipe is configured by the operation pipe portiona and the recovery pipe portion b and prepared in a state that the pipeportion a and the pipe portion b are connected. In one hollow pipe, thenecessary aqueous liquid and gel are filled so as to be alternatelymulti-layered in a necessary order from the lower closed end and therebythe operation medium can be formed, and the operation pipe can bemanufactured. In this case, after a predetermined amount of volume ofthe aqueous liquid in contact with the lower closed end is filled,filling is performed while the vertical thickness of the aqueous liquidlayer and the gel layer multi-layered on the aqueous liquid layer, thatis, the length in the longitudinal direction of the hollow pipe isspecified. When the hollow pipe is configured by the operation pipeportion a and the recovery pipe portion b, first, the recovery unit B iscompleted when filling of the recovery medium necessary for constitutingthe recovery portion B, that is, filling of the aqueous liquid having apredetermined volume or formation of multilayers of the aqueous liquidhaving a predetermined volume and the gel layer having a predeterminedthickness is completed. Furthermore, the operation portion A iscompleted by filling of the operation medium necessary for constitutingthe operation portion A, that is, formation of multilayers of theaqueous liquid layer and the gel layer that have a predeterminedthickness is completed. A more specific method for forming multilayersby alternately multi-layering the aqueous liquid and the gel can beappropriately performed by those skilled in the art according to themulti-layer method in a case of 4-2 described later. Besides, afternecessary aqueous liquid and/or gel are/is filled, the sample supplyportion that is an upper open end may be appropriately closed.

[4-2. A Case in which a Plurality of Hollow Pipes are Prepared forManufacturing One Operation Pipe]

The case in which this creation method is performed may be a case inwhich the pipe is configured by the operation pipe portion a and therecovery pipe portion b and prepared in a state that the pipe portion ais independent from the pipe portion b. In this case, the operationportion A and the recovery portion B are separately manufactured byfilling necessary aqueous liquid and/or gel in each of the pipe portiona and the pipe portion b, and the manufactured operation portion A andrecovery portion B are connected to each other, and thereby theoperation pipe can be manufactured.

An outline of the manufacturing method of the operation portion A isschematically shown in FIGS. 2A to 2C. An aqueous liquid L (for example,a cleaning liquid) constituting the aqueous liquid layer is filled inthe container, and a gel G constituting the gel layer is filled inanother container in a sol state. In FIGS. 2A to 2C, the sol state ismaintained by heating in a constant temperature bath 21 of 70° C. forexample. The lower open end of the pipe portion a is prepared in a stateof being closed by being pressed against a holding mat 22.

A system for feeding liquid into the pipe portion a includes tubes 23and 23′ that respectively extend from the container filled with theaqueous liquid L and the sol-gel G and feed the aqueous liquid L or thesol-gel G, a liquid feeding part 24 (peristaltic pump in FIGS. 2A to 2C)to which the tube 23′ is connected, and a needle 25 for filling the pipeportion a with liquid substances that are fed by the liquid feedingpart. The needle 25 preferably has a length enough to reach thebottommost portion of the pipe portion a by being inserted into the pipeportion a.

In FIGS. 2A to 2C, the tube 23 extending from the container filled withthe aqueous liquid L and the tube 23′ extending from the containercontaining the sol-gel G are connected to a switching valve 26. In thiscase, by switching the valve 26, different liquid substances (theaqueous liquid L and the sol-gel G) can be respectively fed to the sametube 23′ and the same needle 25. This aspect is preferably used when theinner diameter of the pipe portion a is relatively small because onlyone needle is inserted into the pipe portion a.

On the other hand, all liquid feeding paths from the container to theneedle may be made independent without using the switching valve 26. Forexample, when the same operation portion A as FIGS. 2A to 2C ismanufactured, two liquid feeding paths can be formed, one formed by atube extending from the container filled with the aqueous liquid and aneedle connected to the tube and the other formed by a tube extendingfrom the container containing the sol-gel and a needle connected to thetube. In this aspect, the operation pipe is preferably used when theinner diameter of the pipe portion a is relatively large because twoneedles can be inserted into the pipe portion a.

As shown in order in FIGS. 2A-2C, the sol-gel G and the aqueous liquid Lare alternately fed and filled into the pipe portion a in order from thesol-gel G. The leading end of the needle 25 is raised as the liquidlevel in the pipe portion a rises. When the aqueous liquid L ismulti-layered as shown in FIG. 2B after the sol-gel is filled, thepreviously filled sol-gel may be completely gelled or may not becompletely gelled. Usually, the liquid substances that are fed from thecontainer filled with the sol-gel G may be in an intermediate state ofgel-sol with increased viscoelasticity so as to be away from heat source(the constant temperature bath 21 in FIGS. 2A to 2C) when dischargedinto the pipe portion a from the needle 25 inserted into the pipeportion a. Therefore, when the aqueous liquid is multi-layered, even ifthe previous layer 2 is not completely gelled, the contact resistance ofthe gel 2 g against the inner wall of the pipe portion a works, and thegel 2 g having a low specific gravity does not float up. Accordingly, byalternately feeding the sol-gel G and the aqueous liquid L into the pipeportion a, the required number of layers can be formed and the operationportion A can be obtained. Besides, during the filling, the aqueousliquid and the gel are filled with a prescribed thickness based on thethickness of the hollow pipe in the vertical direction.

The recovery portion B can be obtained by filling necessary aqueousliquid or gel. Alternatively, the recovery portion B can be obtained byforming a single layer of the aqueous liquid layer or multilayers of theaqueous liquid layer and the gel layer in an appropriate and necessaryorder in the same manner as described above except that a holding mat isnot used. During the filling, the aqueous liquid is filled with apredetermined amount on the basis of volume, and the gel is filled witha prescribed thickness on the basis of the thickness of the hollow pipein the vertical direction.

The operation portion A and the recovery portion B obtained as describedabove are connected to each other. The operation portion A may beconnected to the recovery portion B in a state that the pipe portion ais inclined and the holding mat is removed and inclined or overturned sothat the contents of the operation portion A do not slide down. As aform of connection, the pipe portion a and the pipe portion b may bewound around a tape or the like, or the pipe portion a and the pipeportion b in which connection portions that can be connected to eachother are respectively formed may be used to connect both of theconnection portions.

Besides, after necessary aqueous liquid and/or gel are/is filled, thesample supply portion which is an upper open end of the operation pipeportion a may be appropriately closed. The timing for closing may beafter the operation portion A is manufactured and before the operationportion A and the recovery portion B are connected, or after theoperation portion A and the recovery portion B are connected.

[5. Magnetic Particles]

The magnetic particles are used to move, by the variations of themagnetic field from outside of the operation pipe, the target componentsin the operation pipe by being accompanied by a small amount ofaccompanying liquid mass. The magnetic particles intended to enableseparation, recovery and purification of specific components by theabove movement usually have chemical functional groups on the surfacesthereof. The magnetic particles may not be filled in the operation pipein advance (FIGS. 1A and 1B) or may be filled in advance (FIG. 1C, FIGS.3A to 3O and FIGS. 4A to 4H). When filled in the operation pipe inadvance, the magnetic particles can be added to the aqueous liquidconstituting the uppermost layer. When the magnetic particles are notadded to the operation pipe in advance, the magnetic particles are alsosupplied to the operation pipe when the sample having the targetcomponents is supplied to the operation pipe.

The magnetic particles are not particularly limited as long as they areparticles that respond to magnetism, and include, for example, particleshaving a magnetic body such as magnetite, γ-iron oxide, manganese zincferrite or the like. In addition, the magnetic particles have a chemicalstructure that specifically binds to the target components supplied tothe above treatment or reaction, and may have a surface containing, forexample, amino group, carboxyl group, epoxy group, avidin, biotin,digoxigenin, protein A, protein G, complex metal ion or antibody, or mayhave a surface that specifically binds to the target components by anelectrostatic force or a van der Waals force. Accordingly, the targetcomponents supplied to the reaction or treatment can be selectivelyadsorbed to the magnetic particles. Hydrophilic groups on the surfacesof the magnetic particles include hydroxyl groups, amino groups,carboxyl groups, phosphoric acid groups, sulfonic acid groups, and thelike.

In addition to the above particles, the magnetic particles can furtherinclude various elements appropriately selected by those skilled in theart. For example, specific forms of the magnetic particles havinghydrophilic groups on the surfaces preferably include particlesconsisting of mixture of magnetic bodies and silica and/or an anionexchange resin, magnetic particles of which the surfaces are covered bythe silica and/or the anion exchange resin, magnetic particles of whichthe surfaces are covered by gold and which have hydrophilic groups viamercapto groups, gold particles containing magnetic bodies and havinghydrophilic groups via mercapto groups on the surfaces, and the like.

As for the size of the magnetic particles having hydrophilic groups onthe surfaces, the average particle size is about 0.1 μm-500 μm. When theaverage particle size is small, the magnetic particles are easy to existin a dispersed state when released from the magnetic field in theaqueous liquid layer. An example of commercially available magneticparticles includes Magnetic Beads which are component reagents ofPlasmid DNA Purification Kit MagExtractor-Plasmid-sold by Toyo Tamotsuand are silica-coated for nucleic acid extraction. When sold as a kit ofcomponent reagents in this way, a product stock solution containingmagnetic particles contains a preservative solution and the like, andthus is preferably cleaned by being suspended in pure water (forexample, about 10 times of the amount). The cleaning can be performed bysuspending in pure water and then removing the supernatant bycentrifugation or aggregation using a magnet, and can be performed byrepeating the suspension and supernatant removal. Besides, the magneticfield applying part for giving magnetic field variations to move themagnetic particles is described in detail in item 8 below.

[6. Method for Operating Target Components in Pipe]

The operations of the target components in the operation pipe are shownin FIGS. 3A-3O and FIGS. 4A-4H. Hereinafter, description will be givenwith reference to FIGS. 3A to 3O and FIGS. 4A-4H.

[6-1. Sample Supply to Operation Pipe]

When the operation pipe is used, a sample 32 containing the targetcomponents is supplied from a reagent supply port 5 (FIG. 3B and FIG.4B). Usually, the sample is supplied in the form of liquid. The samplesupply may be performed manually by a syringe or the like, or may beautomatically controlled by a dispenser using a pipetter or the like.The sample supply is performed in a state that the operation pipe isheld up by an appropriate holding part (not shown; the holding part forholding the operation pipe is described in detail in item 7 below).

In the uppermost layer in the operation pipe, an aqueous liquid mixture33 that contains the sample 32 containing the target components, themagnetic particles 6 and the aqueous liquid are obtained. Morespecifically, the aqueous liquid mixture can be obtained as follows. Forexample, when the uppermost layer filled in the operation pipe consistsof an aqueous liquid, the sample may be supplied into the operation pipetogether with the magnetic particles, or the sample may be supplied intothe operation pipe together with the aqueous liquid and suspendedmagnetic particles. In this way, the aqueous liquid mixture can beobtained from the aqueous liquid in the uppermost layer. In addition,for example, when the uppermost layer filled in the operation pipeconsists of an aqueous liquid containing magnetic particles (the caseillustrated in FIGS. 3A to 3O and 4A to 4H is applicable), the sampleonly may be supplied into the operation pipe, or the sample may besupplied into the operation pipe together with the aqueous liquid. Inthis way, the aqueous liquid mixture can be obtained from the aqueousliquid containing magnetic particles in the uppermost layer.Furthermore, for example, when the uppermost layer filled in theoperation pipe consists of gel, the sample may be supplied into theoperation pipe together with the aqueous liquid and the magneticparticles. In this way, the aqueous liquid mixture can be newly formedas the uppermost layer on the gel layer.

[6-2. Operations in Operation Pipe]

The operation pipe in which the sample is supplied and the aqueousliquid mixture containing the sample and magnetic particles is preparedin the uppermost layer is held up on the holding part and directly seton a device or set on a device under the condition of being transferredto a dedicated holding part in the device. In the device, a magneticfield is generated by bringing a magnetic field applying part (forexample, a cylindrical neodymium magnet having a diameter of 1 mm-5 mmand a length of 5 mm-30 mm) 31 from outside close to an operation pipe1, and magnetic particles 6 dispersed in an aqueous liquid mixture layer31 ₁ are aggregated together with the target components (FIG. 3C andFIG. 4C). At this time, unnecessary components contained in the aqueousliquid mixture layer 31 ₁ can be also aggregated together. By moving themagnetic field applying part 31 downward at a speed of 0.5 mm-10 mm persecond, the magnetic particles accompanied by the target components aretransported from the aqueous liquid mixture layer 31 ₁ via a gel layer 3g ₁ located below and in contact with the aqueous liquid mixture layer31 ₁ (see FIG. 3D and FIG. 4D) to an aqueous liquid layer 31 ₂ locatedbelow and in contact with the gel layer 3 g ₁ (FIG. 3E and FIG. 4E).Besides, since the magnetic particles passing through the gel layer 3 g₁ are thinly coated on the aqueous liquid mixture of the aqueous liquidmixture layer 31 ₁ supplied before passage, the magnetic particles areaccompanied by concomitants in addition to the target components and theconcentration is low. The magnetic particles are further transported tothe aqueous liquid layer 31 ₂. The size and movement speed of the magnetare appropriately determined by those skilled in the art correspondingto the amount of the magnetic particles, the inner and outer diametersof the operation pipe, the state of the gel plug, and the like.

Furthermore, transportation from the aqueous liquid layer 31 ₂ toanother aqueous liquid layer via the gel layer is repeated by themagnetic field applying part 31 as necessary. “Repeating as necessary”means that, as a general rule, the transport operation may be performedas many as the number of times corresponding to the layer number bymoving the magnetic particles only in one direction from the top to thebottom (FIGS. 3E-3N and FIGS. 4E-4H), or the transport operation may beperformed for the number of times above the number corresponding to thelayer number by moving the magnetic particles not only in one directionfrom the top to the bottom but also from the bottom back to the top asappropriate. That is, other aqueous liquid layers of the transportdestination may be present above or below the aqueous liquid layer ofthe transport source. By repeating this transport operation, most of thecontaminants transported by the magnetic particles together with thetarget components are removed. Although the magnetic particlesaccompanying the target components are accompanied by a very smallamount of cleaning liquid, the target components on the surfaces of theparticles are purified to such a degree that a subsequent analysisprocess and the like are not interfered. Accordingly, the purificationof the target components can be performed very efficiently by themagnetic field operation only.

In addition, in the aqueous liquid layer, from the viewpoint ofimproving the processing efficiency, it is preferable to operate so thatthe magnetic particles with the target components (specifically,including target components accompanied by unnecessary components andtarget components from which unnecessary components are removed) can besufficiently brought into contact with the aqueous liquid. As one of themethods for more efficiently performing this operation, there is amethod in which the magnetic field applying part is moved up and down ina state that the magnetic particles are aggregated due to application ofthe magnetic field in the aqueous liquid layer. Other methods includethe method in which the magnetic particles that are aggregated due tothe application of the magnetic field are naturally diffused in theaqueous liquid layer by opening the magnetic field from the magneticparticles that are subjected to the application of the magnetic fieldusing the magnetic field applying part.

As a specific example, as shown in FIG. 3E, the magnetic field isblocked or attenuated by temporarily bringing the magnetic fieldapplying part 31 away from the operation pipe 1, and the magneticparticles are dispersed in a cleaning liquid layer 31 ₂. In this way,the target components adsorbed on the magnetic particles are cleaned bybeing sufficiently exposed in the cleaning liquid 31 ₂ together with theaccompanying components. As shown in FIG. 3F, the magnetic fieldapplying part 31 is brought closer to the operation pipe 1 again andthereby the magnetic particles are aggregated together with the targetcomponents and are in a transportable state. By further moving themagnetic field applying part 31 downward, the magnetic particles aretransported to the gel layer 3 g ₂ just below as shown in FIG. 3G. Inthe magnetic particles and the target components in the gel layer 3 g ₂in FIG. 3G, compared with a case of the magnetic layer particles and thetarget components in the gel layer 3 g ₁ in FIG. 3D, some or most of theaccompanying components are removed by the cleaning in FIG. 3E.

After separating a target substance from the magnetic particles in thelayer filled in the recovery portion B, the magnetic particles fromwhich the target substance is separated are moved from the layer inwhich the target substance is separated to another layer (for example,FIGS. 3N-3O), and thereby the target substance can be recovered in astate of being eluted from the magnetic particles in the recoveryportion.

[6-3. Nucleic Acid Extraction]

For example, when the magnetic particle surfaces are coated with silica,as shown in FIGS. 3A to 3O, the biological sample is supplied to a celllysate 31 ₁ containing a surfactant and a chaotropic salt such asguanidine thiocyanate, and thereby the nucleic acids are liberated fromthe cells (FIG. 3B). The liberated nucleic acids can be specificallyadsorbed on the silica surfaces of the particles. The adsorbed nucleicacids contain reaction inhibition components in this state and thuscannot be utilized as a template for gene amplification reaction.Therefore, the magnetic particles are cleaned by the cleaning liquid 31₂ while the nucleic acids are adsorbed on the surfaces. At this time, inorder to prevent a large amount of reaction inhibition components frombeing introduced into the cleaning liquid, the magnetic particles 6 arecollected by a magnet 31 (FIG. 3C), and are made to pass through a gelplug 3 g ₁ that separates the cell lysate 31 ₁ and the cleaning liquid31 ₂ (FIG. 3D). The magnetic particles can reach the cleaning liquid 31₂ with little liquid fraction when passing through the gel plug 3 g ₁(FIG. 3E). Therefore, the cleaning of the magnetic particles can beimplemented with high efficiency. By further repeating passage throughgel plugs (3 g ₂, 3 g ₃) and transport to cleaning liquids (31 ₃, 31 ₄)of the magnetic particles (FIGS. 3F-3K), purity of the nucleic acids canbe increased. The nucleic acids purified in a state of being adsorbed onthe magnetic particle surfaces are collected again by a magnet (FIG.3L), made to pass through a gel plug 2 g (FIG. 3M), and transported intoan elution liquid 4 (FIG. 3N). In the elution liquid 4, the nucleicacids are separated from the magnetic particles and eluted in theelution liquid. When it is not desired to mix the magnetic particles,the magnetic particles from which the nucleic acids are eluted areretained in the gel plug 2 g again, and the eluted purified nucleicacids remain in the recovery portion B (FIG. 3O). The nucleic acidsobtained in this way are useful as template nucleic acids that can beanalyzed by nucleic acid amplification reaction. The obtained nucleicacids can be used for the next operation (process for performinganalysis by nucleic acid amplification reaction) by removing therecovery portion B of the operation pipe from the operation portion A.

[6-4. Nucleic Acid Synthesis and Analysis]

As shown in FIGS. 4A to 4H, when the operation pipe is used in which thepipe portion a of the operation portion A and the pipe portion b of therecovery portion B are formed integrally, and which has the operationportion A similar to the operation portion in FIGS. 3A to 3O and therecovery portion B filled with a RT reaction liquid 411 and a PCRreaction liquid 412 separated by a gel plug 4 g, after the sameoperation (FIGS. 4B-4F) as that in FIGS. 3B-3M is performed, themagnetic particles 6 are transported to the RT reaction liquid 411 whileadsorbing the purified nucleic acid (RNA) and the RT reaction isperformed (FIG. 4H). After completion of the RT reaction, the magneticparticles also adsorb the DNA (which is a template for PCR reaction)obtained by the RT reaction, pass through the gel plug 4 g to betransported to the PCR reaction liquid 412, and the PCR reaction isperformed (FIG. 4H). The PCR product can be analyzed by a real-timedetection method using a fluorescent dye or a fluorescence detectionmethod using an endpoint detection method. Besides, in FIGS. 4A to 4H,42 and 43 schematically show temperature control functions. A morespecific example of the temperature control function of 42 is shown inthe subsequent item 8-2-6, and a more specific example of thetemperature control function of 43 is shown in the subsequent item 7-3.

When the above operation is performed simultaneously in a plurality ofoperation pipes, multiple channels can be made as shown in FIG. 5. Thedevice illustrated in FIG. 5 has a simple configuration in which amagnetic field applying part (movable magnet plate 53) having a magnetmoving mechanism and a holding substrate with temperature controlfunction (temperature control block 51) are main units. Eachconfiguration is described in items 7 and 8 described later.

[6-5. Protein Synthesis, Separation and Analysis]

[6-5-1. Protein Synthesis Using Hydrogel (P-Gel)]

A cell-free protein synthesis system on the basis ofpolydimethylsiloxane is published in the aforementioned referenceliterature (Nature materials 8, 432-437, 2009). The cell-free proteinsynthesis system is performed in a general-purpose sample tube, but thiscell-free protein synthesis system can also be constructed in theoperation pipe of the disclosure.

[6-5-2. Analysis Using Interaction Between Target Protein and OtherProteins]

A separation and recovery part of protein already exists as acommercially available purification kit, the separation and recoverypart utilizing an antigen-antibody reaction of a protein and an antibody(also a protein) that is manufactured using the protein as a target. Theseparation and recovery are implemented by a protocol using ageneral-purpose tube and a centrifuge. In the cell-free protein systemdescribed above, a spin column is also used separately from the sampletube for separation of the synthesized protein. In the disclosure, byadopting magnetic particles in which the antibody of the target proteinis immobilized on the surface, the target protein can be separated andacquired in one operation pipe without moving the target protein betweendifferent devices.

[6-5-3. Mass Spectrometry in a State that Protein is Adsorbed onMagnetic Particles]

A method for adsorbing a separately prepared protein to be subjected tomass spectrometry to magnetic particles coated with titanium oxide onthe surface, mixing the protein with a matrix in the above state, andanalyzing the protein with a mass spectrometer is described in thereference literature (Analytical Chemistry, 77, 5912-5919, 2005). In thedisclosure, the preparation of the protein to be subjected to massspectrometry and the adsorption to the magnetic particles can beperformed in one operation pipe.

[7. Holding Part]

The operation pipe is usually installed in a substantially verticalshape (that is, in a hold-up state) so that the sample supply portionwhich is an opening portion is on the upper side during use. Anappropriate holding part can be used for installation. In addition, thesame holding part may be used during sample supply and during operationof the target components, or different holding parts may be used. Whendifferent holding parts are used during sample supply and duringoperation of the target components, the transfer of the operation pipebetween the holding parts may be manually performed or be automated.

[7-1. Holding Form]

The holding part is not particularly limited as long as it can be heldin a substantially vertical state (that is, in a hold-up state) so thatthe sample supply portion which is an opening portion of the operationpipe is generally on the upper side. The holding part includes, forexample, a rack which is configured by combining one or two or moreholding members formed with holding holes that can hold the closed endof the operation pipe by piercing of the closed end, or configured byassembling linear members in a lattice shape to form lattice holes asholding holes, but the disclosure is not limited hereto. In the formercase, the holding hole formed in the holding member may penetrate or maynot penetrate the holding member. The inner diameter of the holding holeis determined based on the outer diameter of the operation pipe to beheld. Among the holding members, the one that holds the closed end ofthe operation pipe is described as a holding substrate. In the holdingsubstrate, the holding hole can be formed so that the closed end of theholding portion B does not penetrate the holding substrate (that is, theholding hole itself does not penetrate the holding substrate). The depthof the holding hole is appropriately determined based on the range to beheld in the operation pipe.

[7-2. Holding of a Plurality of Operation Pipes]

Since the operation pipe of the disclosure is elongated and has anextremely small installation area of one pipe during hold-up, aplurality of operation pipes can be held up and installed in aconcentrated state even with a small installation area. As a result, theplurality of operation pipes can be operated simultaneously. That is, amulti-channel operation can be achieved.

An example of this form is shown in FIG. 5. In the device of FIG. 5, upto 20 operation pipes can be processed simultaneously. More operationpipes can also be processed depending on the device specifications. Forexample, if there is an installation area as large as a standard 96-wellplate, up to 96 operation pipes can be held up, and thereby up to 96specimens can be processed simultaneously. In addition, since theoperation pipes are independent from each other, in the above example,the number of operation pipes can be arbitrarily adjusted correspondingto the number of specimens. This form is particularly useful in POCT(Point Of Care Testing) applications in which the number of specimens issmall and the number is also not constant.

When a plurality of operation pipes are held up and installed, forexample, as shown by 51 in FIG. 5, a plurality of holding holes 52 canbe formed in the holding substrate. The holding holes differ dependingon a form of densifying the operation pipes, and can be formed, forexample, in an array shape (that is, formed one-dimensionally in a row)or in a matrix shape (that is, formed two-dimensionally) as shown inFIG. 5. An interval between the holding holes 52 can be appropriatelydetermined based on the density of the operation pipes. When theoperation pipe held by the holding hole 52 has a larger inner diameterin the sample supply portion, the interval between the holding holes 52can be appropriately determined based on the outer diameter of thesample supply portion.

[7-3. Temperature Control Function]

The holding part may have a temperature control function. Morespecifically, the holding part may have a temperature control functionin a portion that holds at least a part of the recovery portion B. Forexample, in FIGS. 4A to 4H, the temperature control function isschematically shown as 43. More specifically, when the holding substrateholds the closed end of the recovery portion B in the holding hole, theholding substrate can have the temperature control function in theportion for holding. For example, a holding substrate 51 shown in FIG. 5holds the closed end of the recovery portion B in a holding hole 52, butthe holding substrate 51 itself may be formed of a temperature controlblock. The temperature control function makes it possible to perform atreatment or reaction requiring temperature control in the aqueousliquid filled at least at the lower end of the recovery portion B. Inthe disclosure, this form is preferably used, for example, when thenucleic acid amplification reaction is performed in the recovery portionB.

[7-4. Optical Detection Port]

The holding substrate may have an optical detection port. The opticaldetection port is arranged to be capable of irradiating an excitationlight into the recovery portion B, and detect a signal derived from thetarget components or components related thereto which is emitted in thetreatment or reaction in the recovery portion B. For example, as shownin FIGS. 7A to 7C, the optical detection port 71 can be formed topenetrate the holding substrate from the lower end of the holding hole52 and has an aperture smaller than the outer diameter of the pipeportion b held by the holding hole 52. An optical detection part(including a fluorescence detection lens 44 and an optical fiber cable45 in FIGS. 7A to 7C) may be arranged in the optical detection port 71.The position of the optical detection port is not limited to theposition in FIGS. 7A to 7C, and for example, photometry from the sidesurface of the recovery portion may be considered.

[8. Magnetic Field Applying Part]

The magnetic field applying part and the magnetic field moving mechanismthereof that cause variations of the magnetic field for moving themagnetic particles in the operation pipe together with the targetcomponents are not particularly limited. As the magnetic field applyingpart, a magnetic source such as a permanent magnet (for example, aferrite magnet or a neodymium magnet) or an electromagnet can be used.Outside the operation pipe, the magnetic field applying part can bedisposed close to the operation pipe to such a degree that it ispossible to aggregate the magnetic particles dispersed in the aqueousliquid layer in the operation pipe on the transport surface side of thepipe and transport the magnetic particles that are aggregated in the gellayer in the operation pipe. Accordingly, the magnetic field applyingpart can effectively generate a magnetic field for the magneticparticles via the transport surface of the pipe, and capture andtransport the target components together with the magnetic particlemass.

[8-1. Shape]

The shape of the magnetic field applying part is not particularlylimited. For example, it may be a massive magnetic field applying partthat can generate a magnetic field at one point or a part of theoperation pipe (for example, illustrated as the magnet 31 in FIGS. 3A to3O or FIGS. 4A to 4H). More specifically, the magnetic field applyingpart may be cylindrical (for example, a diameter of 1 mm-5 mm, athickness of 5 mm-30 mm). In the case of this shape, the magnetic fieldapplying part can generating a magnetic field inside the operation pipeby being attached to one point or a part of the outer periphery of theoperation pipe. On the other hand, the magnetic field applying part maybe a ring-shaped magnet having a substantially-circular-centre hole andcapable of generating a magnetic field around the operation pipe havinga substantially circular cross section. In the case of this shape, themagnetic field applying part can generate a magnetic field inside theoperation pipe by making the operation pipe pass through thesubstantially-circular-centre hole of the ring. In this case, since themagnetic field applying part having a ring shape surrounds the operationpipe, the magnetic particles also have a ring shape according to theshape of the magnetic field applying part when the magnetic particlesare aggregated. On the other hand, if the shape of the magnetic fieldapplying part is a massive shape, the aggregation shape of the magneticparticles is also a massive shape. In other words, when the magneticfield applying part having a ring shape is used, it is preferable interms that a contact area between the magnetic particles and the aqueousliquid is larger and thus the target components and the like adsorbed onthe magnetic particles can be more efficiently exposed in the liquidconstituting the aqueous liquid layer.

[8-2. Magnetic Field Moving Mechanism]

[8-2-1. Movement in Longitudinal Direction of Control Pipe]

The magnetic field moving mechanism of the magnetic field applying partcan move, for example, the magnetic field in the longitudinal direction(axial direction, at least downward direction) of the operation pipe ina state that the aggregation form of the magnetic particles can bemaintained. When described as a magnetic field moving mechanism below,the mechanism can determine the stop position and control the movingspeed, and the control may be performed manually or may be performedautomatically by a computer and the like. The moving speed may be, forexample, 0.5 mm-10 mm per second. The magnetic field moving mechanism ispreferably a mechanism that can physically move the magnetic fieldapplying part itself in the longitudinal direction of the operationpipe. The magnetic field moving mechanism can move the magnetic fieldapplying part (permanent magnet 31 in FIGS. 3A to 3O and FIGS. 4A to 4H)itself as shown in FIGS. 3A to 3O and FIGS. 4A to 4H in the verticaldirection. In addition, even in a device capable of concentrating aplurality of operation pipes as shown in FIG. 5, the magnetic fieldapplying part (movable magnet plate 53 in FIG. 5) can be moved in thevertical direction (the magnetic field moving mechanism itself is notshown in any of the above cases).

[8-2-2. Control of Magnetic Field Intensity]

The magnetic field moving mechanism of the magnetic field applying partmay be a mechanism that can variably control the intensity of themagnetic field applied to the magnetic particles. Specifically, themagnetic field can be blocked or attenuated. The degree of blocking orattenuation of the magnetic field is preferably a degree at which theaggregated magnetic particles can be dispersed in the droplet (the aboveitem 6-2). For example, in the case of an electromagnet, an energizationcontrol part can be used to block the magnetic field. In addition, forexample, in the case of a permanent magnet, a mechanism that can move amagnet disposed outside the operation pipe away from the operation pipecan be used. This mechanism may be controlled manually or automatically.The magnetic particles can be naturally dispersed in the aqueous liquidlayer by attenuating the magnetic field applied to the magneticparticles, preferably by releasing the magnetic particles from themagnetic field. Accordingly, the target components or the accompanyingcomponents adsorbed on the magnetic particles can be sufficientlyexposed in the liquid constituting the aqueous liquid layer.

[8-2-3. A Case of Device in which a Plurality of Control Pipes isConcentrated]

As illustrated in FIG. 5, in a device in which a plurality of operationpipes 1 is concentrated, a plurality of magnetic sources correspondingto the plurality of operation pipes can be held by being unitized intoone member that can move in the longitudinal direction of the operationpipe. As illustrated in FIG. 5, this unitized member can be embodied asthe movable magnet plate 53 which is a magnetic field applying part thatcan move in the longitudinal direction of the operation pipe 1. Asillustrated in FIG. 6, the movable magnet plate 53 in FIG. 5 includes amovable substrate that can move in the longitudinal direction of theoperation pipe and a magnetic source (magnet 31) held in the movablesubstrate, and can be held in a state that a plurality of magnets 31corresponding to each of the operation pipes is disposed. In addition,the member may or may not have a function of holding the operation pipeas the holding part described above. In the case illustrated in FIG. 5,a holding hole 54 corresponding to the operation pipe 1 is formed andthereby the movable magnet plate 53 can also have a holding function. Inthe illustration of FIG. 6, the magnetic field applying part is shown asa massive part, but the magnetic field applying part may have a ringshape being hollow corresponding to the holding hole 54.

As illustrated in FIG. 5, in the device in which the plurality ofoperation pipes 1 is concentrated, the magnetic field moving mechanismof the magnetic field applying part may be capable of simultaneouslycontrolling the intensity of the magnetic field obtained by the magneticfield applying part in each of the plurality of operation pipes. Forexample, when a plurality of different magnetic field applying parts isused for each of the plurality of operation pipes, the magnetic fieldmoving mechanism may be capable of simultaneously controlling themagnetic fields generated by the plurality of magnetic field applyingparts.

In this member, when an electromagnet is used as the magnetic fieldapplying part, the magnetic field can be controlled by current control.On the other hand, when a permanent magnet is used as the magnetic fieldapplying part, in the above member, for example, it is possible toprovide a mechanism that brings the member itself closer to or away fromthe operation pipe (for example, the member itself is movedsubstantially perpendicular to the longitudinal direction of theoperation pipe), or inserts a magnetic shield material therebetween, orbrings the plurality of magnetic field applying parts held in the membercloser to or away from the operation pipe at a time without moving themember itself.

As illustrated in FIG. 6, the movable magnet plate 53 in FIG. 5 can befilled in a magnet holding portion 61 in a state that the magnet 31corresponding to each operation pipe held in the holding hole 54 isdisposed. The magnet holding portion 61 is formed in a size that allowsthe movement of the magnet 31 in the movable magnet plate 53 (that is, amovement of bringing the magnet 31 closer to or away from the operationpipe). As illustrated in FIG. 6, a plurality of magnets 31 can beconnected to each other by connection rods 62, and all the connectionrods 62 can be coupled to a handle member 63. By moving the handlemember 63, as shown in FIG. 6, it is possible to bring all the magnetscloser to the operation pipe (magnetic field applying state) and awayfrom the operation pipe (magnetic field release state).

When the magnet is ring-shaped and this magnet is used to control theintensity of the magnetic field, for example, the magnet that isconfigured by two or more arc-shaped magnet parts and thereby formedinto a ring shape can be used as the ring-shaped magnet. Thisring-shaped magnet can release the operation pipe from the magneticfield by being divided substantially perpendicular to the diameterdirection.

[8-2-4. Movement of Magnetic Field Applying Part in Holding Part Capableof Holding Recovery Portion B]

The holding part may have a recess in which the magnetic field applyingpart can move in the longitudinal direction of the pipe portion b. Morespecifically, the holding part may have, in a portion holding therecovery portion B, a recess in which the magnetic field applying partcan move in the longitudinal direction of the pipe portion b. Themagnetic field applying part that moves in the recess may be the same asor different from the magnetic field applying part contributing to theoperation in the operation portion A. For example, as shown in FIG. 7A,a recess 72 is formed in the holding substrate 51 (in FIGS. 7A to 7C,the holding substrate 51 is configured by a temperature control block)equipped with the holding hole 52, and the recess is filled with amagnet 31′ in advance. The movable magnet plate 53 on which the magnet31 is disposed descends, and as shown in FIG. 7B, the movable magnetplate 53 is in contact with the holding substrate 51 and cannot movefurther downward. That is, depending on the magnet 31, the magneticparticles 6 cannot be transported further downward. At this time, themagnet 31′ filled in the recess 72 of the holding substrate 51 isattracted to the magnet 31 by the magnetic field exerted by the magnet31 on the movable magnet plate 53. Then, the magnetic particles 6 in theoperation pipe 1 are attracted to both the magnet 31 and the magnet 31′.Next, as shown in FIG. 7C, when the magnet 31 in the movable magnetplate 53 is moved away from the operation pipe 1, the magnet 31′ isreleased from the magnetic field generated by the magnet 31 and thusfalls into the recess 72 due to gravity. At this time, the magneticparticles in the operation pipe 1 can be transported into the aqueousliquid 412 in the recovery portion B and be lowered near the bottom inthe recovery portion B due to effects of the magnetic field of themagnet 31′. Accordingly, the magnetic particles can be delivered by themagnet 31 and the magnet 31′, and the magnetic particles accompanied bythe target components can be sufficiently exposed in the lowermost layerin the operation pipe.

[8-2-5. Magnetic Field Fluctuations]

The magnetic field moving mechanism may include a mechanism that enablesa fluctuation motion such as an amplitude movement and rotation of themagnetic field. For example, it is possible to substitute a stirrer byproviding a function that enables the magnetic force source to performan amplitude motion (vertical motion) in the longitudinal direction ofthe operation pipe. Thereby, mixing or stirring in the aqueous liquid isfacilitated. For example, in a case without the function of blocking orattenuating the magnetic field, the magnetic field applying part is madeto reciprocate in the vertical direction for several times within thewidth of the thickness of the aqueous liquid layer while being keptclose to the operation pipe (while the magnetic particles areaggregated), and thereby the target components and the like adsorbed onthe magnetic particles in the aqueous liquid can also be sufficientlyexposed in the liquid constituting the aqueous liquid layer.

[8-2-6. Temperature Control Function]

The magnetic field applying part may further have a temperature controlfunction. For example, in FIGS. 4A to 4H, the temperature controlfunction is schematically shown as 42. Alternatively, a heater can beincorporated in the magnetic field applying part. By the lattertemperature control function, the reagent temperature in the aqueousliquid layer at the position where the magnetic particles are presentcan be arbitrarily adjusted. For example, a case is described in whichthe operation pipe shown in FIGS. 4A to 4H is held by the holding part(holding substrate) as shown in FIGS. 7A to 7C and having thetemperature control function as described in the above 7-3. In theoperation pipe shown in FIGS. 4A to 4H, the recovery portion B fillsmultilayers including the RT reaction liquid layer 411 and the PCRreaction liquid layer 412 via the gel layer 4 g as a recovery medium.When the operation pipe of FIGS. 4A to 4H is held by the holdingsubstrate 51 as shown in FIGS. 7A to 7C, the portion directly held inthe holding hole 52 of the holding substrate 51 may be onlyapproximately a portion corresponding to the lowest layer of theoperation pipe (PCR reaction liquid layer 412). In this case, since theportion filled with the RT reaction liquid layer 411 in which thereverse transcription reaction is performed is separated from the PCRreaction liquid layer 412 held directly on the holding substrate 51, itis difficult to add temperature control using the holding substrate 51.

Therefore, the device of the disclosure can have a temperature controlfunction different from the temperature control function in the holdingpart. For example, as illustrated as 42 in FIGS. 4A to 4H, thetemperature control function may not be interlocked with the magneticfield applying part; alternatively, as illustrated as 64 in FIG. 6, thetemperature control function may be incorporated in the movable magnetplate 53 which is a magnetic field applying part and thereby beinterlocked with the magnetic field applying part. In a specific aspectof the movable magnet plate 53 shown in FIG. 6 in which the temperaturecontrol function (heater) is incorporated, a heater 64 has an annularshape enclosing the holding hole 54. When the movable magnet plate 53has the temperature control function in this way, in a period in whichthe movable magnet plate 53 is in a position filled with the RT reactionliquid layer 411 (FIG. 7A), the RT reaction liquid layer 411 is heatedby the heater 64 in the movable magnet plate 53 and the optimumtemperature (for example, 50° C.) can be achieved.

[9. Optical Detection Part]

The optical detection part is not particularly limited and can be easilyselected by those skilled in the art corresponding to the analysismethod in which the target components are supplied. For example, a partcan be used which appropriately includes a light generation portion, adetection part, a light transmission part, a personal computer and thelike. For example, in a case of the fluorescence detection part 41 shownin FIG. 4H, as shown more specifically in FIGS. 7A to 7C, incidence fromthe light generation portion (not shown) to the detection part (thelight transmission part attached to a detection lens 44 (the opticalfiber cable 45)) is performed, and light irradiation to the reactionliquid 4 in the operation pipe 1 through the detection lens 44 can beperformed. The optical signal detected by the detection lens 44 can besent to a light receiving element by the optical fiber cable 45,converted into an electrical signal, and then transmitted in real timeto a personal computer (not shown), and changes in the fluorescenceintensity of the reaction liquid 4 can be monitored. This is suitablewhen the disclosure performs a reaction or treatment such as a real-timenucleic acid amplification reaction in which a variable fluorescenceintensity is detected.

An LED, a laser, a lamp or the like can be used as the light generationportion. In addition, in the detection, various light receiving elementsfrom inexpensive photodiodes to photomultiplier tubes aiming at highersensitivity can be utilized without particular limitation. For example,when a case in which a nucleic acid-related reaction such as a real-timenucleic acid amplification reaction or a nucleic acid-related treatmentis performed is used as an example, for example, when SYBR (registeredtrademark) GREEN I is used, this dye is specifically bound to adouble-stranded DNA and generates fluorescence around 525 nm, and thusthe detection part can detect a light having a target wavelength bycutting lights having wavelengths other than the target wavelength withan optical filter. In addition, when a case in which the nucleic acidamplification reaction using droplet movement is performed in theoperation pipe is used as an example, the fluorescence observation ofthe droplet supplied to the nucleic acid amplification reaction can beperformed in a darkroom in a state that excitation lights are irradiatedto a temperature position in which an extension reaction (usually about68-74° C.) using DNA polymerase is performed and the liquid droplet isstopped in this position. Furthermore, when an irradiation range of theexcitation light is expanded from a temperature position in which heatdenaturation is performed to a temperature position in which annealingis performed, the droplet can be moved and a melting curve of theamplification products can also be obtained.

EXAMPLE

Next, examples are given to describe the disclosure in more detail, butthe scope of the disclosure is not limited hereto.

Example 1

[Nucleic acid extraction and purification from blood]A gelling agent(Taiyo Chemical Co., Ltd., TAISET 26) is added to silicon oil (Shin-EtsuSilicone KF-56) to reach a ratio of 1.2% (weight ratio) and heated to70° C. to be completely mixed with the silicon oil. A required amount ofthe oil mixed into a sol state and a required amount of necessaryreagents are alternately injected and multi-layered from the tip of theinjection needle into the operation pipe (consisting of a capillary(operation portion A) and a sample tube (recovery portion B)) shown inFIG. 3A in a manner that bubbles do not enter. When the capillary havingan inner diameter of 1.5 mm is used, respectively 10 μL of the gel plug,15 μL of the cleaning liquid (200 mM of KCl), and 20 μL of the elutionliquid (10 mM of TrisHCl, 1 mM of EDTA pH 8.0)) are filled as shown inFIG. 3A. The filled capillary is placed at room temperature for 30minutes to completely gel the gel plug. The upper end of the capillaryforms a funnel-shaped sample supply port that is sealed by a filmmaterial and is sealed by a septum.

The uppermost layer in the capillary is made into 100 μL of a celllysate (4M guanidine thiocyanate, 2% (w/v) of Triton X-100, and 100 mMof Tris-HCl pH 6.3) and contains 500 μg of silica-coated magneticparticles (nucleic acid extraction kit, MagExtractor-Plasmid-attachedmagnetic particles of Toyobo). Besides, as a nucleic acid isolationmethod using silica particles and chaotropic salts, a method disclosedby Boom et al. (Japanese Patent Laid-Open No. 2-289596) is used.

FIGS. 3B-3O are diagrams showing a nucleic acid extraction process fromblood for each operation of the magnet. Finally, the nucleic acid isrecovered in the elution liquid in the sample tube attached to the lowerend of the capillary. In FIG. 3B, 200 μL of whole human blood isinjected by an injection needle and is gently mixed with the magneticparticles by pipetting. After five minutes, as shown in FIGS. 3C and 3D,the magnetic particles are collected by bringing the magnet close fromone side of the capillary, and the magnet is lowered at a speed of 0.5mm per second. After the magnetic particles pass through the gel plug,as shown in FIG. 3E, the magnet is separated from the capillary. Asshown in FIGS. 3F-3M, the same cleaning is performed for three times.Thereafter, the magnet is released as shown in FIG. 3N, and the magneticparticles are dropped into the tube containing the elution liquid. Afterone minute, the magnet is brought close again to collect the magneticparticles, and as shown in FIG. 3O, the magnet particles are retractedinto the gel plug, and the nucleic acid extraction and purificationoperations are completed. In this example, 200 ng of DNA is obtained for1 μL of the elution liquid.

The sample tube is removed from the capillary, 1 μL of the elutionliquid obtained in the sample tube is used, and a PCR reaction mixture(10 μL of total reaction volume) containing 0.15 U of Taq DNApolymerase, 500 nM of human GAPDH gene detectionprimer(5′-GCGCTGCCAAGGCTGTGGGCAAGG-3′ (Sequence number 1) and5′-GGCCCTCCGACGCCTGCTTCACCA-3′ (Sequence number 2)) and 200 nM of dNTPis used to perform PCR (temperature cycle: 95° C., one second, 60° C.,ten seconds, 72° C., ten seconds, 40 cycles) by a thermal cycler(ABI9700, Applied Biosystems). As a result, as shown in FIG. 8, areaction product specific to the human GAPDH gene (fragment size 171bases) is confirmed by agarose gel electrophoresis.

As described above, the disclosure is described according to theembodiments of the disclosure, but it should not be considered that thedescription and drawings constituting a part of this disclosure limitthe disclosure. From this disclosure, various alternative embodiments,examples, and operational techniques are apparent to those skilled inthe art. The technical scope of the disclosure is defined only by theinvention specific matters of the scope of claims reasonable from theabove description, and can be modified and embodied without departingfrom the scope in the implementation stage.

Sequence numbers 1 and 2 are synthetic primers.

What is claimed is:
 1. An operation pipe for operating targetcomponents, comprising: a hollow pipe, having a closable open end forsupplying a sample containing the target components on one side and aclosed end on the other side, and having an operation pipe portion onthe open end side and a recovery pipe portion on the closed end side; anoperation medium, which is filled in the operation pipe portion so thatgel layers and aqueous liquid layers are alternately multi-layered inthe longitudinal direction of the hollow pipe, wherein a layer length ofthe gel layers and a layer length of the aqueous liquid layers aredetermined by the length in the longitudinal direction of the hollowpipe; a recovery medium, which is filled in the recovery pipe portion sothat a gel layer and an aqueous liquid layer which is in contact withthe closed end are multi-layered, wherein the aqueous liquid layer incontact with the closed end has a predetermined volume, and the layerlength of the gel layer is determined by the length in the longitudinaldirection of the hollow pipe; and magnetic particles for capturing andtransporting the target components; wherein the magnetic particles passthrough the gel layer in a gel state and move in the longitudinaldirection of the operation pipe due to application of a magnetic field.2. The operation pipe according to claim 1, wherein an inner diameter ofthe hollow pipe is 0.1 mm-5 mm.
 3. The operation pipe according to claim1, wherein a volume of the aqueous liquid layer in contact with theclosed end is 1 μL-1000 μL.
 4. The operation pipe according to claim 1,wherein the operation pipe portion and the recovery pipe portion areseparable.
 5. The operation pipe according to claim 1, wherein thematerial of the hollow pipe is selected from a group consisting ofpolyethylene, polypropylene, fluororesin, polyvinyl chloride,polystyrene, polycarbonate, acrylonitrile-butadiene-styrene copolymer,acrylonitrile-styrene copolymer, acrylic resin, polyvinyl acetate,polyethylene terephthalate, cyclic polyolefin, and glass.
 6. Theoperation pipe according to claim 1, wherein an inner diameter of theopen end is larger than an inner diameter of the operation pipe portionand an inner diameter of the recovery pipe portion.
 7. The operationpipe according to claim 1, wherein the hollow pipe has opticaltransparency.
 8. The operation pipe according to claim 1, whereinsurface roughness of an inner surface of the hollow pipe is 0.1 Lm orless.
 9. The operation pipe according to claim 1, wherein a length ofthe gel layer in the longitudinal direction of the hollow pipe is 1-20mm, the gel layer being filled in the operation pipe portion and therecovery pipe portion.
 10. The operation pipe according to claim 1,wherein a length of the aqueous liquid layer in the longitudinaldirection of the hollow pipe is 0.5-30 mm, the aqueous liquid layerbeing filled in the operation pipe portion.
 11. The operation pipeaccording to claim 1, wherein the magnetic particles are particleshaving a binding force or adsorption force to nucleic acids that areused as the target components, the aqueous liquid layer in the operationmedium is an aqueous liquid layer containing a liquid that liberatesnucleic acids and binds or adsorbs the nucleic acids to the magneticparticles and/or an aqueous liquid layer containing a cleaning liquid ofthe magnetic particles, and the aqueous liquid layer in the recoverymedium which is in contact with the closed end is an aqueous liquidlayer containing a liquid that liberates nucleic acids.
 12. Theoperation pipe according to claim 11, wherein the aqueous liquid layerin the recovery medium which is in contact with the closed end is anaqueous liquid layer further containing a reverse transcription reactionliquid and/or a nucleic acid amplification reaction liquid.
 13. Theoperation pipe according to claim 12, wherein the aqueous liquid layerin the recovery medium which is in contact with the closed end is anaqueous liquid layer further containing a fluorescent dye that is usedto be specifically bound to the target components and detect the targetcomponents by generating fluorescence by light irradiation.
 14. Adevice, comprising: the operation pipe according to claim 13; a magneticfield applying part, which is capable of moving the magnetic particlesin the longitudinal direction of the operation pipe by applying amagnetic field to the operation pipe; and an optical detection part,which irradiates light to the recovery pipe portion and detectsfluorescence generated from the fluorescent dye specifically bound tothe target components.
 15. A device comprising a plurality of operationpipes according to claim 1, and further comprising a magnetic fieldapplying part capable of simultaneously moving, for the plurality ofoperation pipes, the magnetic particles in the longitudinal direction ofthe operation pipe by simultaneously applying a magnetic field to theplurality of operation pipes.
 16. The device according to claim 15,wherein the magnetic field applying part comprises a movable substratecapable of moving in the longitudinal direction of the operation pipe; amagnetic field moving mechanism, which controls movement of the movablesubstrate toward the longitudinal direction of the operation pipe; and aplurality of magnetic sources, which corresponds to the plurality ofoperation pipes and is held in the movable substrate.
 17. A devicecomprising the operation pipe according to claim 1, and a magnetic fieldapplying part capable of moving the magnetic particles in thelongitudinal direction of the operation pipe by applying a magneticfield to the operation pipe; wherein the magnetic field applying partcauses the magnetic field to perform amplitude movement in thelongitudinal direction of the operation pipe or causes the magneticfield to perform rotational motion.